Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines.

In Alzheimer disease (AD) the microtubule-associated protein tau is redistributed exponentially into paired helical filaments (PHFs) forming neurofibrillary tangles, which correlate with pyramidal cell destruction and dementia. Amorphous neuronal deposits and PHFs in AD are characterized by aggregation through the repeat domain and C-terminal truncation at Glu-391 by endogenous proteases. We show that a similar proteolytically stable complex can be generated in vitro following the self-aggregation of tau protein through a high-affinity binding site in the repeat domain. Once started, tau capture can be propagated by seeding the further accumulation of truncated tau in the presence of proteases. We have identified a nonneuroleptic phenothiazine previously used in man (methylene blue, MB), which reverses the proteolytic stability of protease-resistant PHFs by blocking the tau-tau binding interaction through the repeat domain. Although MB is inhibitory at a higher concentration than may be achieved clinically, the tau-tau binding assay was used to identify desmethyl derivatives of MB that have Ki values in the nanomolar range. Neuroleptic phenothiazines are inactive. Tau aggregation inhibitors do not affect the tau-tubulin interaction, which also occurs through the repeat domain. Our findings demonstrate that biologically selective pharmaceutical agents could be developed to facilitate the proteolytic degradation of tau aggregates and prevent the further propagation of tau capture in AD.

Secretory granule targeting of atrial natriuretic peptide correlates with its calcium-mediated aggregation.

Atrial natriuretic peptide (ANP) is a 28-aa peptide hormone secreted predominantly from atrial cardiocytes. ANP is first synthesized in the form of a 126-aa precursor (proANP) which is targeted to dense core granules of the regulated secretory pathway. ProANP is stored until the cell receives a signal that triggers the processing and release of the mature peptide (regulated secretion). Various models have been proposed to explain the targeting of selected proteins to the regulated secretory pathway, including specific "sorting receptors" and calcium-mediated aggregation. As potential calcium binding regions had previously been reported in the profragment of ANP, the current study was undertaken in an effort to determine the relationship between the ability of ANP to enter the regulated secretory pathway and its calcium-mediated aggregation. Deletion and site-directed mutagenesis of selected regions of the prosegment demonstrates that acidic amino acids at positions 23 and 24 are critical for both regulated secretion of proANP from transfected AtT-20 cells and calcium-mediated aggregation of purified recombinant proANP in vitro. These results demonstrate that the ability of certain proteins to enter secretory granules is directly linked to their calcium-mediated aggregation.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population.

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

Identification and expression analysis of a potential familial Alzheimer disease gene on chromosome 1 related to AD3.

The inheritance of much early-onset Alzheimer disease (AD) has been linked to a dominant-acting locus on chromosome 14. Recently, the gene likely responsible for this genetic linkage has been identified and termed AD3. Five mutations have been found in AD3 that segregate with the disease phenotype in seven AD families and are not present in unaffected individuals. Here we report the existence of a gene encoding a seven transmembrane domain protein very similar to that encoded by AD3 in structure and sequence. This gene is located on chromosome 1, is expressed in a variety of tissues, including brain, and is predicted to harbor mutations causing nonchromosome 14 familial AD. The presence of several S/TPXX DNA binding motifs in both the AD3 protein and the AD3-like protein /AD4 protein suggests a possible role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane. Ways in which mutations in either gene could lead to AD are discussed.

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease amyloid, binds A beta and stimulates A beta aggregation.

NACP, a 140-amino acid presynaptic protein, is the precursor of NAC [the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease (AD) amyloid], a peptide isolated from and immunologically localized to brain amyloid of patients afflicted with AD. NACP produced in Escherichia coli bound to A beta peptides, the major component of AD amyloid. NACP bound to A beta 1-38 and A beta 25-35 immobilized on nitrocellulose but did not bind to A beta 1-28 on the filter under the same conditions. NACP binding to A beta 1-38 was abolished by addition of A beta 25-35 but not by A beta 1-28, suggesting that the hydrophobic region of the A beta peptide is critical to this binding. NACP-112, a shorter splice variant of NACP containing the NAC sequence, bound to A beta, but NACP delta, a deletion mutant of NACP lacking the NAC domain, did not bind A beta 1-38. Furthermore, binding between NACP-112 and A beta 1-38 was decreased by addition of peptide Y, a peptide that covers the last 15 residues of NAC. In an aqueous solution, A beta 1-38 aggregation was observed when NACP was also present in an incubation mixture at a ratio of 1:125 (NACP/A beta), whereas A beta 1-38 alone or NACP alone did not aggregate under the same conditions, suggesting that the formation of a complex between A beta and NACP may promote aggregation of A beta. Thus, NACP can bind A beta peptides through the specific sequence and can promote A beta aggregation, raising the possibility that NACP may play a role in the development of AD amyloid.

De novo biosynthesis of the aggregation pheromone components ipsenol and ipsdienol by the pine bark beetles Ips paraconfusus Lanier and Ips pini (Say) (Coleoptera: Scolytidae).

The California five-spined ips, Ips paraconfusus Lanier, produces the myrcene-derived acyclic monoterpene alcohols ipsenol (2-methyl-6-methylene-7-octen-4-ol) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) as components of its aggregation pheromone. The pine engraver beetle, Ips pini (Say), produces only ipsdienol. Previous studies have shown that myrcene, a monoterpene in the pines colonized by these beetles, is a direct precursor to these pheromone components. In vivo radiolabeling studies reported here showed that male I. paraconfusus incorporated [1-14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol), while male I. pini incorporated [1-14C]acetate into ipsdienol and amitinol. Females of these species produced neither labeled nor unlabeled pheromone components. The purified radiolabeled monoterpene alcohols from-males were identified by comparison of their HPLC and GC retention times with those of unlabeled standards. HPLC-purified fractions containing the individual radiolabeled components were analyzed by GC-MS and were shown to include only the pure alcohols. To further confirm that ipsdienol and ipsenol were radiolabeled, diastereomeric ester derivatives of the isolated alcohols were synthesized and analyzed by HPLC and GC-MS. After derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring stereochemistry. The results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles.

A mutation in the promoter of the lipoprotein lipase (LPL) gene in a patient with familial combined hyperlipidemia and low LPL activity.

We have identified a naturally occurring mutation in the promoter of the lipoprotein lipase (LPL) gene. One of 20 patients with familial combined hyperlipidemia (FCHL) and reduced levels of postheparin plasma LPL activity was found to be a heterozygote carrier of this mutation. The mutation, a T-->C substitution at nt -39, occurred in the binding site of the transcription factor Oct-1. As a result, the transcriptional activity of the mutant promoter was < 15% of wild type, as determined by transfection studies in the human macrophage-like cell line THP-1. This decrease in promoter activity was observed in undifferentiated as well as in phorbol ester-differentiated THP-1 cells. Furthermore, the inductive effect of elevating the levels of intracellular cAMP was equally reduced. This mutation was not present among 20 FCHL patients with normal plasma LPL levels nor has it been reported among individuals with familial LPL deficiency. Thus, heterozygosity for LPL promoter mutations may be one of several factors that contribute to the etiology of FCHL.

Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy.

In 10-30% of hypertrophic cardiomyopathy kindreds, the disease is caused by > 29 missense mutations in the cardiac beta-myosin heavy chain (MYH7) gene. The amino acid sequence similarity between chicken skeletal muscle and human beta-cardiac myosin and the three-dimensional structure of the chicken skeletal muscle myosin head have provided the opportunity to examine the structural consequences of these naturally occurring mutations in human beta-cardiac myosin. This study demonstrates that the mutations are related to distinct structural and functional domains. Twenty-four are clustered around four specific locations in the myosin head that are (i) associated with the actin binding interface, (ii) around the nucleotide binding site, (iii) adjacent to the region that connects the two reactive cysteine residues, and (iv) in close proximity to the interface of the heavy chain with the essential light chain. The remaining five mutations are in the myosin rod. The locations of these mutations provide insight into the way they impair the functioning of this molecular motor and also into the mechanism of energy transduction.

Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

Expansible Computable Aggregation Rules

The aggregation operator have been considered from a computable point of view. The important condition that the computation is friendly when portions of data are inserted o deleted to the list of values to aggregate is considered.

Mutações inativadoras no gene MKRN3 são causa de puberdade precoce central familial

A maioria dos casos de puberdade precoce central (PPC) em meninas permanece idiopática. A hipótese de uma causa genética vem se fortalecendo após a descoberta de alguns genes associados a este fenótipo, sobretudo aqueles implicados com o sistema kisspeptina (KISS1 e KISS1R). Entretanto, apenas casos isolados de PPC foram relacionados à mutação na kisspeptina ou em seu receptor. Até recentemente, a maioria dos estudos genéticos em PPC buscava genes candidatos selecionados com base em modelos animais, análise genética de pacientes com hipogonadismo hipogonadotrófico, ou ainda, nos estudos de associação ampla do genoma. Neste trabalho, foi utilizado o sequenciamento exômico global, uma metodologia mais moderna de sequenciamento, para identificar variantes associadas ao fenótipo de PPC. Trinta e seis indivíduos com a forma de PPC familial (19 famílias) e 213 casos aparentemente esporádicos foram inicialmente selecionados. A forma familial foi definida pela presença de mais de um membro afetado na família. DNA genômico foi extraído dos leucócitos do sangue periférico de todos os pacientes. O estudo de sequenciamento exômico global realizado pela técnica ILLUMINA, em 40 membros de 15 famílias com PPC, identificou mutações inativadoras em um único gene, MKRN3, em cinco dessas famílias. Pesquisa de mutação no MKRN3 realizada por sequenciamento direto em duas famílias adicionais (quatro pacientes) identificou duas novas variantes nesse gene. O MKRN3 é um gene de um único éxon, localizado no cromossomo 15 em uma região crítica para a síndrome de Prader Willi. O gene MKRN3 sofre imprinting materno, sendo expresso apenas pelo alelo paterno. A descoberta de mutações em pacientes com PPC familial despertou o interesse para a pesquisa de mutações nesse gene em 213 pacientes com PPC aparentemente esporádica por meio de reação em cadeia de polimerase seguida de purificação enzimática e sequenciamento automático direto (Sanger). Três novas mutações e duas já anteriormente identificadas, incluindo quatro frameshifts e uma variante missense, foram encontradas, em heterozigose, em seis meninas não relacionadas. Todas as novas variantes identificadas estavam ausentes nos bancos de dados (1000 Genomes e Exome Variant Server). O estudo de segregação familial em três dessas meninas com PPC aparentemente esporádica e mutação no MKRN3 confirmou o padrão de herança autossômica dominante com penetrância completa e transmissão exclusiva pelo alelo paterno, demonstrando que esses casos eram, na verdade, também familiares. A maioria das mutações encontradas no MKRN3 era do tipo frameshift ou nonsense, levando a stop códons prematuros e proteínas truncadas e, portanto, confirmando a associação com o fenótipo. As duas mutações missenses (p.Arg365Ser e p.Phe417Ile) identificadas estavam localizadas em regiões de dedo ou anel de zinco, importantes para a função da proteína. Além disso, os estudos in silico dessas duas variantes demonstraram patogenicidade. Todos os pacientes com mutação no MKRN3 apresentavam características clínicas e hormonais típicas de ativação prematura do eixo reprodutivo. A mediana de idade de início da puberdade foi de 6 anos nas meninas (variando de 3 a 6,5) e 8 anos nos meninos (variando de 5,9 a 8,5). Tendo em vista o fenômeno de imprinting, análise de metilação foi também realizada em um subgrupo de 52 pacientes com PPC pela técnica de MS-MLPA, mas não foram encontradas alterações no padrão de metilação. Em conclusão, este trabalho identificou um novo gene associado ao fenótipo de PPC. Atualmente, mutações inativadoras no MKRN3 representam a causa genética mais comum de PPC familial (33%). O MKRN3 é o primeiro gene imprintado associado a distúrbios puberais em humanos. O mecanismo preciso de ação desse gene na regulação da secreção de GnRH necessita de estudos adicionais

Aggregation dynamics and foraging behaviour of striped red mullet Mullus surmuletus in the western Mediterranean Sea

Habitat-related heterogeneity of striped red mullet Mullus surmuletus heterospecific foraging assemblages was examined off the coast of Spain. Video-based focal-follows conducted on 122 M. surmuletus assemblages (446 total individuals) revealed an array of attendant species (n = 7) with composition linked to benthic habitat complexity; bare sandy substrata were characterized by homospecific groups of M. surmuletus, while habitats with rock and vegetation attracted a variety of scrounging labrids and sparids. Although the nature of the relationship between M. surmuletus and attendants requires further exploration, the present study indicates that substratum composition can be a driving factor explaining the dynamics of this heterospecific assemblage.