RESUMO
BACKGROUND: Half of mental health disorders begin before the age of 14, highlighting the importance of prevention and early-intervention in childhood. Schools have been identified globally by policymakers as a platform to support good child mental health; however, the majority of the research is focused on secondary schools, with primary schools receiving very little attention by comparison. The limited available evidence on mental health initiatives in primary schools is hindered by a lack of rigorous evaluation. This quasi-experimental cluster study aims to examine the implementation and effectiveness of a Mental Health and Wellbeing Co-ordinator role designed to build mental health capacity within primary schools. METHODS: This is a primary (ages 5-12) school-based cluster quasi-experimental study in Victoria, Australia. Before baseline data collection, 16 schools selected by the state education department will be allocated to intervention, and another 16 matched schools will continue as 'Business as Usual'. In intervention schools, a mental health and well-being coordinator will be recruited and trained, and three additional school staff will also be selected to receive components of the mental health training. Surveys will be completed by consenting staff (at 2-, 5-, 10- and 17-months post allocation) and by consenting parents/carers (at 3-, 10- and 17-months post allocation) in both intervention and business as usual schools. The primary objective is to assess the change in teacher's confidence to support student mental health and wellbeing using the School Mental Health Self-Efficacy Teacher Survey. Secondary objectives are to assess the indirect impact on systemic factors (level of support, prioritisation of child mental health), parent and teachers' mental health literacy (stigma, knowledge), care access (school engagement with community-based services), and student mental health outcomes. Implementation outcomes (feasibility, acceptability, and fidelity) and costs will also be evaluated. DISCUSSION: The current study will examine the implementation and effectiveness of having a trained Mental Health and Wellbeing Coordinator within primary schools. If the intervention increases teachers' confidence to support student mental health and wellbeing and builds the capacity of primary schools it will improve student mental health provision and inform large-scale mental health service reform. TRIAL REGISTRATION: The trial was retrospectively registered in the Australian New Zealand Clinical Trials Registry (ANZCTR) on July 6, 2021. The registration number is ACTRN12621000873820 .
Assuntos
Saúde Mental , Serviços de Saúde Escolar , Criança , Pré-Escolar , Humanos , Instituições Acadêmicas , Estudantes , VitóriaRESUMO
OBJECTIVES: There is evidence that HIV-positive patients are suffering from a greater burden of morbidity as they age due to nonAIDS-related complications. To date it has been difficult to determine what part of this excess risk is due to the health effects of HIV, its treatment or to lifestyle factors common to gay and bisexual men (GBM). We calculated overall and cause-specific hospitalisation rates and risk factors for hospitalisations in HIV-negative and HIV-positive cohorts of GBM and compare these with rates in the general male population. METHODS: We conducted a record linkage study, linking two cohorts of HIV-negative (n = 1325) and HIV-positive (n = 557) GBM recruited in Sydney, New South Wales (NSW), Australia with the NSW hospital discharge data register. We compared rates of hospitalisation in the two cohorts and risk factors for hospitalisation using random-effects Poisson regression methods. Hospitalisation rates for each cohort were further compared with those in the general male population using indirect standardisation. RESULTS: We observed 2032 hospitalisations in the HIV-negative cohort during 13,016 person-years (PYs) [crude rate: 15.6/100 PYs (95% CI: 14.9-16.3)] and 2130 hospitalisations in the HIV-positive cohort during 5571 PYs [crude rate: 38.2/100 PYs (95% CI: 36.6-39.9)]. HIV-positive individuals had an increased risk of hospitalisation compared with the HIV-negative individuals [adjusted-IRR: 2.34 (95% CI: 1.91-2.86)] and the general population [SHR: 1.45 (95% CI: 1.33-1.59)]. Hospitalisation rates were lower in the HIV-negative cohort compared with the general population [SHR: 0.72 (95% CI: 0.67-0.78)]. The primary causes of hospitalisation differed between groups. CONCLUSIONS: HIV-positive GBM continue to experience excess morbidity compared with HIV-negative GBM men and the general population. HIV-negative GBM had lower morbidity compared with the general male population suggesting that GBM identity does not confer excess risk.
Assuntos
Bissexualidade/estatística & dados numéricos , Infecções por HIV/epidemiologia , Homossexualidade Masculina/estatística & dados numéricos , Hospitalização/estatística & dados numéricos , Adulto , Austrália/epidemiologia , Estudos de Coortes , Comorbidade/tendências , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Fatores de RiscoRESUMO
BACKGROUND: Uncertainty exists about the best treatment for people with HIV-1 who have virological failure with first-line combination antiretroviral therapy of a non-nucleoside analogue (NNRTI) plus two nucleoside or nucleotide analogue reverse transcriptase inhibitors (NtRTI). We compared a second-line regimen combining two new classes of drug with a WHO-recommended regimen. METHODS: We did this 96-week, phase 3b/4, randomised, open-label non-inferiority trial at 37 sites worldwide. Adults with HIV-1 who had confirmed virological failure (plasma viral load >500 copies per mL) after 24 weeks or more of first-line treatment were randomly assigned (1:1) to receive ritonavir-boosted lopinavir plus two or three NtRTIs (control group) or ritonavir-boosted lopinavir plus raltegravir (raltegravir group). The randomisation sequence was computer generated with block randomisation (block size four). Neither participants nor investigators were masked to allocation. The primary endpoint was the proportion of participants with plasma viral load less than 200 copies per mL at 48 weeks in the modified intention-to-treat population, with a non-inferiority margin of 12%. This study is registered with ClinicalTrials.gov, number NCT00931463. FINDINGS: We enrolled 558 patients, of whom 541 (271 in the control group, 270 in the raltegravir group) were included in the primary analysis. At 48 weeks, 219 (81%) patients in the control group compared with 223 (83%) in the raltegravir group met the primary endpoint (difference 1·8%, 95% CI -4·7 to 8·3), fulfilling the criterion for non-inferiority. 993 adverse events occurred in 271 participants in the control group versus 895 in 270 participants in the raltegravir group, the most common being gastrointestinal. INTERPRETATION: The raltegravir regimen was no less efficacious than the standard of care and was safe and well tolerated. This simple NtRTI-free treatment strategy might extend the successful public health approach to management of HIV by providing simple, easy to administer, effective, safe, and tolerable second-line combination antiretroviral therapy. FUNDING: University of New South Wales, Merck, AbbVie, the Foundation for AIDS Research.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , Lopinavir/administração & dosagem , Pirrolidinonas/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Ritonavir/administração & dosagem , Adulto , Quimioterapia Combinada , Feminino , Infecções por HIV/virologia , Inibidores da Protease de HIV/administração & dosagem , HIV-1/efeitos dos fármacos , Humanos , Masculino , Nucleosídeos/administração & dosagem , Nucleotídeos/administração & dosagem , Raltegravir Potássico , Resultado do TratamentoRESUMO
The beta-amyloid precursor protein (beta-APP), which is involved in the pathogenesis of Alzheimer's disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown gamma-secretases. Here we show that an affinity reagent designed to interact with the active site of gamma-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer's and are known mediators of gamma-secretase cleavage of both beta-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of gamma-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer's disease.
Assuntos
Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Marcadores de Afinidade , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases , Células CHO , Cricetinae , Dimerização , Humanos , Proteínas de Membrana/química , Microssomos/química , Microssomos/metabolismo , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Presenilina-2 , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Processamento de Proteína Pós-Traducional , TransfecçãoRESUMO
Polyadenylate [poly(A)] polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs). The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP). Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site. The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides. The first two domains are functionally analogous to polymerase palm and fingers domains. The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer. In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1. One occupies the position of the incoming base, prior to its addition to the mRNA chain. The other is believed to occupy the position of the 3' end of the mRNA primer.
Assuntos
Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/metabolismo , Polinucleotídeo Adenililtransferase/química , Polinucleotídeo Adenililtransferase/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Manganês/metabolismo , Modelos Moleculares , Mutação , Proteínas Associadas a Pancreatite , Polinucleotídeo Adenililtransferase/genética , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteína S6 Ribossômica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismoRESUMO
The cerebro-hepato-renal syndrome is a rare familial malady with cerebral, renal, and skeletal abnormalities, severe hypotonia, cirrhosis, iron and lipid storage, and death within 6 months. Correlated electron microscopic, histochemical, and biochemical studies demonstrate defects in two oxidative organelles. Peroxisomes cannot be found in hepatocytes and renal proximal tubules. In hepatocytes and cortical astrocytes, mitochondria are distorted in their appearance and glycogen stores are increased. Oxygen consumnption of brain and liver mitochondrial preparations with succinate and with substrates reducing nicotinamide adenine dinucleotide is markedly diminished, but the consumption is normal with ascorbate and tetramethylphenylenediamine, which suggests a defect in electron transport prior to the cytochromes. Histochemical studies of mitochondrial oxidation point to a defect between the succinate dehydrogenase flavoprotein and coenzyme Q, possibly in the region of nonheme iron protein.
Assuntos
Doenças Ósseas/patologia , Córtex Cerebral/patologia , Túbulos Renais/patologia , Fígado/patologia , Organoides , Acidose/metabolismo , Acidose/patologia , Doenças Ósseas/metabolismo , Encéfalo/metabolismo , Química Encefálica , Feminino , Humanos , Lactente , Túbulos Renais Proximais/patologia , Lipídeos/análise , Masculino , Microcorpos , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/metabolismo , Músculos/patologia , Consumo de Oxigênio , SíndromeRESUMO
Little is known about the transcriptional events which occur downstream of polyadenylation sites. Although the polyadenylation site of a gene can be easily identified, it has been difficult to determine the site of transcription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach, we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 and GAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of the ADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of the poly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sized nonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. A GAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site. However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site. Mutations of GAL7 or ADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis of the extended poly(A)- transcript. Since transcription of the mutant template continues through this region in vivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data support the hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcription termination.
Assuntos
Poli A/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Regiões Terminadoras Genéticas , Transcrição Gênica , Sistema Livre de Células , Análise Mutacional de DNA , Técnicas In Vitro , Precursores de Ácido Nucleico/metabolismo , RNA Fúngico/metabolismo , Deleção de SequênciaRESUMO
Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1). Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1. We present here the identification of separate functional domains of Fip1. Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity. This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function. Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor. A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1. Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation. In the absence of this domain, Pap1 remains in an inhibited state. These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex.
Assuntos
Sistemas de Transporte de Aminoácidos , Proteínas de Membrana/metabolismo , Polinucleotídeo Adenililtransferase , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Divisão Celular/genética , Sobrevivência Celular , Teste de Complementação Genética , Mutação , Proteínas Associadas a Pancreatite , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNARESUMO
In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.
Assuntos
Regiões 3' não Traduzidas/biossíntese , Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Poli A/metabolismo , Poliadenilação/fisiologia , RNA Mensageiro/biossíntese , Proteínas de Saccharomyces cerevisiae , Substituição de Aminoácidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/genética , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Ligação Proteica/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae , Especificidade por Substrato/fisiologia , Fatores de Poliadenilação e Clivagem de mRNARESUMO
Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.
Assuntos
Poli A/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/isolamento & purificação , Adenovírus Humanos/genética , Sistema Livre de Células , Centrifugação , Células HeLa , Técnicas In Vitro , Substâncias Macromoleculares , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares Pequenas , Relação Estrutura-AtividadeRESUMO
The sequences directing formation of mRNA 3' ends in Saccharomyces cerevisiae are not well defined. This is in contrast to the situation in higher eukaryotes in which the sequence AAUAAA is known to be crucial to proper 3'-end formation. The AAUAAA hexanucleotide is found upstream of the poly(A) site in some but not all yeast genes. One of these is the gene coding for alcohol dehydrogenase, ADH2. Deletion or a double point mutation of the AAUAAA has only a small effect on the efficiency of the reaction, and in contrast to the mammalian system, it is most likely not operating as a major processing signal in the yeast cell. However, we isolated point mutations which reveal that a region located approximately 80 nucleotides upstream of the poly(A) site plays a critical role in either transcription termination, polyadenylation, or both. These mutations represent the first point mutations in yeasts which significantly reduce the efficiency of 3'-end formation.
Assuntos
Álcool Desidrogenase/genética , Mutação , Poli A/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , Genes Fúngicos , Dados de Sequência Molecular , RNA Fúngico/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologiaRESUMO
The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.
Assuntos
Quinases Ciclina-Dependentes , Proteínas Serina-Treonina Quinases/genética , RNA Polimerase II/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae , Transcrição Gênica , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiaeRESUMO
The postnatal development of myelin in the optic nerve and tract of normal and dark reared cats has been studied quantitatively with light and electron microscopy. In the newborn cat few myelinated fibers (3% of the population) are seen in the optic tract. Until the end of the second postnatal week, the total number of myelinated axons in the tract remains low (23%). At this time, however, there is an explosive increase in the rate of myelination and by the end of the fourth postnatal week 80% of the optic tract axons have acquired a myelin sheath. Thereafter, the number of myelinated axons increases gradually, reaching adult levels (100%) at 12 weeks. During the initial period of myelination, the average axon diameter is 0.6 mu for unmyelinated fibers and a.2 mu for myelinated fibers. Both of these means remain substantially unchanged until myelination is completed, suggesting that initial myelination of an axon is not a continuous process but rather proceeds in a step-wise manner. Dark rearing appears to have no effect on the initiation of myelination.
Assuntos
Bainha de Mielina/fisiologia , Nervo Óptico/crescimento & desenvolvimento , Envelhecimento , Animais , Axônios/ultraestrutura , Gatos , Bainha de Mielina/ultraestrutura , Neuroglia/ultraestrutura , Nervo Óptico/ultraestrutura , Estimulação LuminosaRESUMO
Oxidative phosphorylation was measured polarographically in brain mitochondria isolated from 1 to 3-week-old normal and triiodothyronine-treated rat pups. Adenosine diphosphate (ADP)/oxygen ratios with nicotinamide-adenine dinucleotide (NAD)-linked substrates, but not with succinate, were increased in brain mitochondria from experimental animals at each age. Control ratios and respiratory rates were not affected. Thus, the normal maturational increase in ADP/oxygen ratios with NAD-linked substrates is accelerated in brain mitochondria from rats treated with triiodothyronnie from birth. This effect on efficiency of oxidative phosphorylation is similar to that of throid hormones on other properties of the maturing brain.
Assuntos
Encéfalo/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Masculino , Mitocôndrias/metabolismo , NAD/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Succinatos/metabolismoRESUMO
The final step in the generation of the amyloid-beta protein (Abeta), implicated in the etiology of Alzheimer's disease, is proteolysis within the transmembrane region of the amyloid precursor protein (APP) by gamma-secretase. Although considered an important target for therapeutic design, gamma-secretase has been neither well-characterized nor definitively identified. Previous studies in our laboratory using substrate-based difluoro ketone and difluoro alcohol transition-state analogue inhibitors suggest that gamma-secretase is an aspartyl protease with loose sequence specificity. To further characterize the active site of gamma-secretase, we prepared a series of difluoro ketone peptide analogues with varying steric bulkiness in the P1 position and tested the ability of these compounds to inhibit Abeta production in APP-transfected cells. Incorporation of bulky, aliphatic P1 side chains, such as sec-butyl or cyclohexylmethyl, led to increased gamma-secretase inhibitory potency, suggesting a large S1 pocket to accommodate these substituents and providing further evidence for loose sequence specificity. The cyclohexylmethyl P1 substituent allowed N-terminal truncation to a low-molecular-weight compound (<600 Da) that effectively blocked Abeta production (IC(50) approximately 5 microM). This finding suggests that optimal S1 binding may allow the development of potent inhibitors with ideal pharmaceutical properties. Moreover, a difluoro alcohol analogue with a cyclohexylmethyl P1 substituent was equipotent with its difluoro ketone counterpart, providing strong evidence that gamma-secretase is an aspartyl protease. All new analogues inhibited total Abeta and Abeta(42) production with the same rank order of potency and increased Abeta(42) production at low concentrations, providing further evidence for distinct gamma-secretases that are nevertheless closely similar with respect to active site topology and mechanism.
Assuntos
Doença de Alzheimer/enzimologia , Endopeptidases/metabolismo , Cetonas/síntese química , Peptídeos/química , Inibidores de Proteases/síntese química , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Animais , Células CHO , Domínio Catalítico , Linhagem Celular , Cricetinae , Desenho de Fármacos , Cetonas/química , Cetonas/farmacologia , Mimetismo Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/biossíntese , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Relação Estrutura-AtividadeRESUMO
We describe a patient with Hirschsprung disease and autism. High-resolution karyotyping indicated that the patient has an interstitial deletion of 20p11.22-p11.23. Microsatellite analysis showed a deletion involving a 5-6 cM region from the maternally derived chromosome 20. The deleted region is proximal to, and does not overlap, the recently characterized Alagille syndrome region. This region of 20p has not yet been implicated in Hirschsprung disease or autism. However, this region contains several genes that could plausibly contribute to any phenotype that includes abnormal neural development.
Assuntos
Transtorno Autístico/complicações , Transtorno Autístico/genética , Deleção Cromossômica , Cromossomos Humanos Par 20/genética , Doença de Hirschsprung/complicações , Doença de Hirschsprung/genética , Síndrome de Alagille/genética , Criança , Cromossomos Humanos Par 20/ultraestrutura , Feminino , Impressão Genômica , Perda Auditiva/complicações , Perda Auditiva/genética , Humanos , Masculino , FenótipoRESUMO
Both sexual differentiation, which is a matter of individual development, and sexual dissimilation, which is a matter of individual differences, result from developmental processes that are open to input from the early maternal environment. There are reliable features in both the dam and the young that ensure that males receive more perineal stimulation from maternal licking than is necessary for survival and normal growth. This stimulation contributes toward the development of masculine sexual behavior and mechanisms in the central nervous system that control copulatory reflexes. Because of differences in signals that they produce, males receive more stimulation than females. This bias in early stimulation accounts for some of the dissimilarity between the sexes in nervous system morphology and behavior. The same processes that produce sex differences can also produce individual differences among males. These differences are likely to have significant functional consequences in rats, a species in which males have a high level of intrasexual reproductive competition. Future research will be directed toward testing this functional hypothesis and toward exploring the extent of stimulative effects on the development of the sexually dimorphic brain regions that function in sexual behavior.
Assuntos
Vias Aferentes , Biologia do Desenvolvimento , Comportamento Materno , Estimulação Física , Desenvolvimento Psicossexual , Comportamento Sexual Animal , Canal Anal/inervação , Animais , Estudos de Avaliação como Assunto , Genitália/inervação , Ratos , Caracteres Sexuais , LínguaRESUMO
The amyloid-beta protein (A beta), strongly implicated in the etiology of Alzheimer's disease (AD), is formed from the amyloid-beta precursor protein (APP) through sequential proteolysis by beta- and gamma-secretases. Cleavage by gamma-secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40- and 42-amino acid A beta C-terminal variants, A beta 40 and A beta 42. The latter form of A beta is highly fibrillogenic, is invariably elevated in autosomal-dominant forms of AD, and is the major A beta component found presymptomatically in cerebral deposits. Thus, blocking production of A beta in general and A beta 42 in particular is considered an important therapeutic goal. We have developed transition-state analogue inhibitors of gamma-secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of gamma-secretase activity in APP-transfected cells, strongly suggesting an aspartyl protease mechanism; (2) gamma-secretases that form A beta 40 and A beta 42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for gamma-secretases; (4) A beta 42 production is increased several fold over control by these gamma-secretase inhibitors after replacement with inhibitor-free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total A beta and A beta 42 production hours after replacement with compound-free media and should help identify the target(s) of these protease transition-state mimics.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/genética , Animais , Ácido Aspártico Endopeptidases , Sítios de Ligação , Células CHO , Cricetinae , Humanos , Oligopeptídeos/química , Inibidores de Proteases/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/antagonistas & inibidores , TransfecçãoRESUMO
BACKGROUND: Hypoxia and warm ischemia produce severe injury to cardiac grafts harvested from non-heartbeating donors. To potentially improve recovery of such grafts, we studied the effects of intravenous phenylephrine preconditioning. METHODS: Thirty-seven blood-perfused rabbit hearts were studied. Three groups of non-heart-beating donors underwent intravenous treatment with phenylephrine at 12.5 (n = 8), 25 (n = 7), or 50 microg/kg (n = 7) before initiation of apnea. Non-heart-beating controls (n = 8) received saline vehicle. Hypoxic cardiac arrest occurred after 6 to 12 minutes of apnea, followed by 20 minutes of warm in vivo ischemia. A 45-minute period of ex vivo reperfusion ensued. Nonischemic controls (n = 7) were perfused without antecedent hypoxia or ischemia. RESULTS: Phenylephrine 25 microg/kg significantly delayed the onset of hypoxic cardiac arrest compared with saline controls (9.6 +/- 0.5 versus 7.7 +/- 0.4 minutes; p = 0.00001), yet improved recovery of left ventricular developed pressure compared with saline controls (57.1 +/- 5.3 versus 41.0 +/- 3.4 mm Hg; p = 0.04). Phenylephrine 25 microg/kg also yielded a trend toward less myocardial edema than saline vehicle (p = 0.09). CONCLUSIONS: Functional recovery of nonbeating cardiac grafts is improved by preconditioning. We provide evidence that the myocardium can be preconditioned with phenylephrine against hypoxic cardiac arrest.
Assuntos
Parada Cardíaca/cirurgia , Transplante de Coração/métodos , Precondicionamento Isquêmico Miocárdico/métodos , Fenilefrina/administração & dosagem , Análise de Variância , Animais , Água Corporal/metabolismo , Infusões Intravenosas , Isquemia Miocárdica/cirurgia , Reperfusão Miocárdica , Miocárdio/metabolismo , Consumo de Oxigênio , Coelhos , Função Ventricular EsquerdaRESUMO
The role of maternal stimulation in the development of a lumbar motor nucleus (spinal nucleus of the bulboca vernosus, SNB) was investigated. The perineum, which has afferents to the lumbar region, is stimulated throughout early development by maternal licking, a behavior that is elicited by chemosignals secreted by the pups. In the present study, half of the dams were treated with intranasal zinc sulfate throughout the postpartum period, which led to a specific reduction in maternal stimulation of pup perineum by interfering with the reception of eliciting signals. Adult offspring of both sexes from anosmic dams had 11% fewer SNB motor neurons than normally stimulated controls, an effect which was most apparent in the rostral portion of the nucleus. There was no effect of treatment on neuron size. It was concluded that afferent input provided by species-typical maternal behavior contributes to the number of neurons that survive the neonatal period of normal cell death.