Amyloid-β: the seeds of darkness

Whilst the amyloid-β (Aβ) hypothesis/centric theory continues to evolve, genetic, biochemical and pathological evidence still suggests that Aβ is central to the etiology of Alzheimer's disease (AD). In particular, Aβ-oligomers/soluble Aβ, may be an earlier determinant of Alzheimer's disease and better correlative of cognitive impairment. Whilst there are a number of Aβ-oligomeric species in existence (making therapeutic and diagnostic biomarker choice cumbersome), their existence is in equilibrium with Aβ-fibrils, the main constituent of cored plaques. Although Alzheimer's disease remains incurable, improvements to Aβ immunotherapies and strategies to target Aβ continue to evolve, with the reliance upon Aβ imaging to shed light on the outcome of therapeutics proving very useful.     

β-glucanases of the yeast Pichia polymorpha

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl--d-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A₅₀ has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different -glucanases located in the periplasmic space is presented.     

β-thalassemia mutations in the Portuguese population

Summary In this study we have carried out haplotype analysis on the -globin gene cluster and characterized the -thalassemia mutation by oligonucleotide hybridization in 14 patients with thalassemia major and 5 with sickle cell/-thalassemia originating from southern Portugal. We found that three mutations, namely the °-39, ° IVS-1 nt 1 and ⁺ IVS-1 nt 110 are prevalent accounting for 53%, 32% and 10% of the -thalassemia chromosomes respectively. In general each mutation was associated with a specific chromosomal haplotype; the ° mutation, however, was linked to three different haplotypes. These results indicate that three oligo-probes complementary to the most common mutations allow prenatal diagnosis by oligonucleotide analysis in 96% of the couples at risk of having offspring with thalassemia major in southern Portugal.     

β-catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii

The canonical Wnt/β-catenin pathway is a key regulator of body plan organization and axis formation in metazoans, being involved in germ layer specification, posterior growth and patterning of the anteroposterior axis. Results from animals spanning a wide phylogenetic range suggest that a unifying function of β-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by β-catenin has been demonstrated in distantly related animals (cnidarians, a protostome, echinoderms and ascidians), the definition of the posterior part of the embryo is well supported only for vertebrates and planarians. To gain insights into β-catenin functions during deuterostome evolution, we have studied the early development of the direct developing hemichordate Saccoglossus kowalevskii. We show that the zygote is polarized after fertilization along the animal-vegetal axis by cytoplasmic rearrangements resembling the ascidian vegetal contraction. This early asymmetry is translated into nuclear accumulation of β-catenin at the vegetal pole, which is necessary and sufficient to specify endomesoderm. We show that endomesoderm specification is crucial for anteroposterior axis establishment in the ectoderm. The endomesoderm secretes as yet unidentified signals that posteriorize the ectoderm, which would otherwise adopt an anterior fate. Our results point to a conserved function at the base of deuterostomes for β-catenin in germ layer specification and to a causal link in the definition of the posterior part of the embryonic ectoderm by way of activating posteriorizing endomesodermal factors. Consequently, the definition of the vegetal and the posterior regions of the embryo by β-catenin should be distinguished and carefully re-examined.     

On the structure of the human striate area. Lamina IVcβ

Summary Layer IVc of the human striate area consists mainly of a great number of small spinous local circuit neurons which store numerous characteristic lipofuscin granules. Since the neurons of the neighbouring layers are almost devoid of pigment deposits the boundaries of lamina IVc are easily traceable. Hence, the pigment granules can be used as internal markers to unequivocally identify these small pigmented spinous local circuit neurons of lamina IVc in ultrathin sections. They have a large spherical nucleus surrounded by a narrow cytoplasmic rim poor in organelles, and very scarcely receive axosomatic symmetric synapses.Within layer IVc four types of synaptic boutons can be distinguished. Type-1-boutons are large, contain a few and loosely arranged round vesicles and make asymmetric synaptic contacts with dendrites and dendritic spines. The type-2-boutons which are also large are filled with densely packed round vesicles which accumulate at the presynaptic membrane. The large type-3-boutons are characterized by elongated vesicles and symmetric synaptic contact zones. These boutons generate several fingerlike protrusions. Small profiles which contain elongated vesicles and form symmetric synaptic contacts, are most probably parts of these protrusions. The large amount of small boutons with round vesicles and asymmetric synaptic contact zones are tentatively described as type-4-boutons although it is far from certain that they represent a uniform class. The presumable origins of the different types of boutons are discussed.Supported by the Deutsche Forschungsgemeinschaft (Br. 634/1)Dedicated to Prof. Dr. med. H. Leonhardt in honor of his 60th birthday     

β-Decay and the origin of optical purity

Improving Keszthelyis simple model the evolutionary appearance of concentration difference of enanthiomeric compounds due to their differential decomposition by -rays is investigated taking into account the racemization as well. It is shown that if the difference in the cross sections is very small then the resulting concentration difference will never exceed the statistical fluctuations, while in the case of a sufficiently large difference in the cross sections the concentration difference can overgrow the statistical fluctuations in an evolutionary reasonable period of time. The relative difference of the concentrations, however, will be generally much smaller than that of the cross sections. Therefore some other, amplifying mechanism must be postulated in order to explain the optical purity of living beings.     

Characterization of the CaMKKβ-AMPK signaling complex

The AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis, and is a potential target for treatment of metabolic diseases as well as cancer. AMPK can be phosphorylated and activated by the tumor suppressor LKB1 or the Ca²⁺/CaM-dependent protein kinase kinase β (CaMKKβ). We previously identified a physical complex between CaMKKβ and AMPK (Anderson, K. A., Ribar, T. J., Lin, F., Noeldner, P. K., Green, M. F., Muehlbauer, M. J., Witters, L. A., Kemp, B. E., and Means, A. R. (2008) Cell Metabolism 7, 377–388). Here we expand our analysis of the CaMKKβ–AMPK signaling complex and show that whereas CaMKKβ can form a complex with and activate AMPK, CaMKKα cannot. In addition, we show that CaMKKβ and AMPK associate through their kinase domains, and CaMKKβ must be in an active conformation in order to bind AMPK but not to associate with an alternative substrate, Ca²⁺/Calmodulin-dependent protein kinase IV (CaMKIV). Our results demonstrate that CaMKKβ and AMPK form a unique signaling complex. This raises the possibility that the CaMKKβ–AMPK complex can be specifically targeted by small molecule drugs to treat disease.     

β-Catenin mediates the anti-adipogenic effect of baicalin

β-Catenin reportedly inhibits adipogenesis through the down-regulations of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer binding protein (C/EBP)α. We report that baicalin, a natural flavonoid compound, inhibits adipogenesis by modulating β-Catenin. During 3T3-L1 cell adipogenesis, β-Catenin was down-regulated, but baicalin treatment maintained β-Catenin expression. Anti-adipogenic effects of baicalin were significantly attenuated by β-Catenin siRNA transfection. β-Catenin siRNA rescued the reduced expressions of PPARγ, C/EBPα, fatty acid binding protein 4 and lipoprotein lipase by baicalin. Furthermore, baicalin modulated members of the WNT/β-Catenin pathway by maintaining the expressions of low-density lipoprotein receptor-related protein 6, disheveled (DVL)2 and DVL3. These findings suggest that β-Catenin mediates the anti-adipogenic effects of baicalin.     

β-lapachone accelerates the recovery of burn-wound skin

β-lapachone is a quinone of lapachol extracted from the bark of lapacho tree. Recent findings demonstrated that punched skin wounds of mice healed faster with β-lapachone treatment. The present study investigates the effects of β-lapachone on burn-wound skin of C57BL/6 mice injured by a 100 °C iron stick. Our results indicated that wounds treated with β-lapachone recovered faster than those treated with control ointment containing no β-lapachone. On the third day after burning, the area of β-lapachone treated-wound was 30% smaller than wound treated with control ointment. H&E and immunohistochemistry staining showed that burn-wound skin treated with ointment containing β-lapachone healed faster in its epidermis, dermis, and underlying connective tissues with more macrophages appeared than those treated with control ointment alone. RAW264.7 cell, a macrophage-like cell line derived from BALB/C mice, was used as a model for scrutinizing the effect of β-lapachone on macrophages. We found that the proliferation and the secretion of EGF and VEGF by macrophages were higher in cultures treated with β-lapachone and that ?-lapachone can also increase the release of EGF with TNF-α pretreatment. We conclude that β-lapachone plays an important role in accelerating burn wound healing, and that β-lapachone not only can raise the proliferation of macrophages but also increase the release of VEGF from macrophages.     

β-Glucuronidase Mutants of the Nematode CAENORHABDITIS ELEGANS

Using a screening procedure that is based on a histochemical stain for the enzyme beta-glucuronidase, we have isolated several mutants of the nematode Caenorhabditis elegans affected in beta-glucuronidase activity. All of the mutations fall into one complementation group and identify a new gene, gus-1, which has been mapped on the right arm of linkage group I (LG I), 1.1 map units to the left of unc-54. The mutants have no visible phenotype, and their viabilities and fertilities are unaffected. Linked revertants of two of the mutations have been isolated. They restore enzyme activity to almost wild-type levels; the beta-glucuronidase that one of the revertants produces differs from that of the wild type. We propose that gus-1 is the structural locus for beta-glucuronidase.     

On the histochemical demonstration of N-acetyl-β-galactosaminidase

Summary Aging neurons accumulate lipofuscin pigment granules which appear to be secondary lysosomes of the residual body variety. The biological significance of the residual bodies is debated. They were here studied with the aim of testing a hypothesis that the membranes surrounding these granules might be more vulnerable than the membranes around younger types of lysosomes.For this purpose large motor neurons of young and old rats were compared with respect to lysosomal membrane latency, using a modified Bitensky lysosomal lability test. Utilizing successively increasing incubation times, the lysosomes of old neurons, in particular the residual bodies in polar aggregates of old neurons—presumed to represent lipofuscin pigment granules—were found to have a clearly reduced latency in comparison with lysosomes of young neurons.These findings support the notion that the residual bodies are more fragile than younger lysosomes.Supported by the Swedish Medical Research Council (Project No. 12X-2037).     

βIII Spectrin Regulates the Structural Integrity and the Secretory Protein Transport of the Golgi Complex

A spectrin-based cytoskeleton is associated with endomembranes, including the Golgi complex and cytoplasmic vesicles, but its role remains poorly understood. Using new generated antibodies to specific peptide sequences of the human βIII spectrin, we here show its distribution in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network. The use of a drug-inducible enzymatic assay that depletes the Golgi-associated pool of PI4P as well as the expression of PH domains of Golgi proteins that specifically recognize this phosphoinositide both displaced βIII spectrin from the Golgi. However, the interference with actin dynamics using actin toxins did not affect the localization of βIII spectrin to Golgi membranes. Depletion of βIII spectrin using siRNA technology and the microinjection of anti-βIII spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. At ultrastructural level, Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, we show that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in βIII spectrin-depleted cells. However, the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. We state that βIII spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain its structural integrity and secretory activity, and unlike actin, PI4P appears to be highly relevant for the association of βIII spectrin the Golgi complex.     

Beyond the frog: the evolution of homology models of human IKKβ

Nuclear Factor κ B is implicated in tumor progression and chronic inflammatory diseases and is regulated by IκB kinase β (IKKβ). The crystal structure of IKKβ has been recently solved for Xenopus laevis. Homology models of human IKKβ have been developed prior to and after the crystal structure was solved. Here, we compare four models of human IKKβ and evaluate their performance in both broad and focused library docking studies.     

NMR structures of the transmembrane domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.     

β-Spectrin Regulates the Hippo Signaling Pathway and Modulates the Basal Actin Network

Emerging evidence suggests functional regulation of the Hippo pathway by the actin cytoskeleton, although the detailed molecular mechanism remains incomplete. In a genetic screen, we identified a requirement for β-Spectrin in the posterior follicle cells for the oocyte repolarization process during Drosophila mid-oogenesis. β-spectrin mutations lead to loss of Hippo signaling activity in the follicle cells. A similar reduction of Hippo signaling activity was observed after β-Spectrin knockdown in mammalian cells. We further demonstrated that β-spectrin mutations disrupt the basal actin network in follicle cells. The abnormal stress fiber-like actin structure on the basal side of follicle cells provides a likely link between the β-spectrin mutations and the loss of the Hippo signaling activity phenotype.     

β-Globin gene polymorphism in the Saudi Arab population

Summary DNA mapping with the restriction endonucleases, Hpa I and Mst II, has been used to investigate -globin gene polymorphism in the Saudi Arab population. Using Hpa I digestion, 13.0kb and 7.6kb fragments were found in association with the ^A and ^S genes. The frequency of the polymorphic forms in two regions investigated vary significantly. In Al-Hafouf and the surrounding villages, situated in the Eastern Province of Saudi Arabia, the frequencies of association of the ^S gene with the Hpa I 7.6kb, 7.0kb, and 13.0kb fragments were 0.866, 0.043, and 0.071, respectively. The frequency of association of ^A with the 7.6kb and 13.0kb fragments resulting from the Hpa I digestion were 0.875 and 0.125. In Khaiber, Tehamat-Aseer, and surrounding villages, in the Western Province, the frequency of association of ^S with 7.6kb, 7.0kb, and 13.0kb fragments were 0.836, 0.027, and 0.0136, respectively, while that of ^S was 0.250 and 0.750 with 7.0kb and 13.0kb Hpa I fragments, respectively. Using Mst II digestion, ^A was found to be linked to a 1.15kb fragment, while ^s was linked to a 1.35kb fragment. The normal (Hb AA), heterozygotes (Hb AS), and homozygotes (Hb SS) gave 1.15, 1.15/1.35, and 1.35kb fragments, respectively. The results of this study show extensive polymorphism at the Hpa I restriction site of the ^A and ^S globin genes with the different polymorphic forms existing at a variable frequency in different regions of Saudi Arabia.     

Leucyl-β-naphthylamide-splitting enzymes in the mammalian endocrine pancreas

The proteolytic capacity of the endocrine pancreas in obese-hyperglycaemic mice was evaluated by using the chromogenic substrate l-leucyl-beta-naphthylamide (LNA). The analytical sensitivity obtained with this substrate in photometric and fluorimetric assays permitted quantitative determinations of C-N-bond-splitting activity in both crude islet homogenates and electrophoretic fractions thereof. The following observations were made: (1) The rate of LNA cleavage was maximal at about pH7 in islets as well as in acinar tissue. An apparent K(m) of 4x10(-5)-6x10(-5)m was calculated for both the endocrine and exocrine pancreas. (2) The level of LNA-splitting enzyme activity was of the same magnitude in the islets as in the liver, and significantly higher in the islets than in the exocrine pancreas. Starving the animals for 7 days did not affect the enzyme activity levels. (3) Two distinct LNA-splitting enzymes could be separated from the pancreatic islets by means of disc electrophoresis, the most rapidly migrating band representing the highest activity. Though similar electrophoresis patterns were obtained with acinar tissue and liver, only one enzyme could be demonstrated in serum. The data suggest that the beta-cells, in addition to being highly specialized for the production of a specific protein, contain a comparatively high capacity for protein catabolism.     

GARP regulates the bioavailability and activation of TGFβ

Glycoprotein-A repetitions predominant protein (GARP) associates with latent transforming growth factor-β (proTGFβ) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFβ activation and the structural basis of coassociation remain unknown. We find that Cys-192 and Cys-331 of GARP disulfide link to the TGFβ1 prodomain and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFβ1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFβ-binding protein for binding to proTGFβ1. Association between GARP and proTGFβ1 prevents the secretion of TGFβ1. Integrin α(V)β(6) and to a lesser extent α(V)β(8) are able to activate TGFβ from the GARP-proTGFβ1 complex. Activation requires the RGD motif of latent TGFβ, disulfide linkage between GARP and latent TGFβ, and membrane association of GARP. Our results show that GARP is a latent TGFβ-binding protein that functions in regulating the bioavailability and activation of TGFβ.