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Vitamin D is a critical fat-soluble vitamin for the nervous system. Research suggests a potential link between vitamin D deficiency and attention-deficit hyperactivity disorder (ADHD), particularly in children and adolescents. The core symptoms of ADHD are associated with deficits in striatal functions, and maintaining sufficient levels of vitamin D may help prevent or alleviate ADHD symptoms. However, the molecular changes in the striatum caused by vitamin D supplementation that may contribute to the brain processes linked to ADHD symptoms remain unclear. In this study, we established a mouse model fed diets with three different dose gradients of vitamin D3 (0, 500, and 2000 IU/kg·day) from postnatal day 21 (P21) to 14 weeks of age. Striatal tissues from mice with gradient vitamin D3 intake were subjected to reduced representation bisulfite sequencing (RRBS), RNA-sequencing, and neurotransmitter profiling by liquid chromatography-mass spectrometry (LC-MS). Our findings indicate that vitamin D supplementation since childhood influenced the overall landscape of DNA methylations and the expression of many genes involved in critical neurological functions in a dose-dependent manner. Additionally, our data demonstrate how vitamin D modulated neuropeptide signaling pathways, as well as cholinergic and dopaminergic synapses in the striatum, through an orchestrated mechanism involving epigenetic and transcriptional regulations. Furthermore, we observed a synergistic effect of vitamin D on dopamine release following acute methylphenidate injection into our mouse model. In summary, this study provides mechanistic insights into how dietary vitamin D supplementation since childhood can modulate specific signal transductions among striatal cells, underscoring the importance of vitamin D supplementation for ADHD management.
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Objective: To understand the prevalence of Chlamydia trachomatis (CT) infection and its associated factors among asymptomatic outpatients attending sexually transmitted disease (STD)-related clinics in Shenzhen and provide evidence for development of future interventions. Methods: From April 15 to May 16, 2018, a cross-sectional study was conducted and patients attending STD-related Clinics were recruited from 22 medical institutions in Nanshan, Luohu, Bao'an, Longgang, Yantian, and Longhua districts of Shenzhen. After the informed consent from each participant was obtained, social-demographic information was collected through a structured questionnaire and urine samples were collected for CT nucleic acid detection. In addition, logistic regression was used to explore associated factors of CT infection. Results: In asymptomatic outpatients, the prevalence of CT infection was 7.16% (250/3 492). Being single (aOR=2.29, 95%CI:1.65-3.16), without registered Shenzhen residency (aOR=1.49, 95%CI:1.04-2.13), and without previous CT testing in the past year (aOR=2.04, 95%CI:1.03-4.05) were the risk factors of CT infection in asymptomatic outpatients. Among participants without registered Shenzhen residency, 89.25% (2 176/2 438) were college-degree or below, and 51.29% (1 255/2 447) were aged ≤30 years, and the risk of CT infection among those ≤30 years old was 1.73 times higher than those >30 years old (95%CI:1.28-2.34). Conclusions: The prevalence of CT infection was high among asymptomatic outpatients attending STD-related clinics in Shenzhen. Routine CT screening should be carried out for this population, especially for those with sexually active age, being single, with low educational level, and without previous CT testing in the past year. Also, raising their awareness of knowledge and adverse outcomes of CT infection should be considered to promote routine CT screening and timely treatment.
Assuntos
Infecções por Chlamydia , Ácidos Nucleicos , Adulto , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis , Estudos Transversais , Humanos , Pacientes AmbulatoriaisRESUMO
Objective: To evaluate the influence of duodenal stump reinforcing on the short-term complications after laparoscopic radical gastrectomy. Methods: A retrospective cohort study with propensity score matching (PSM) was conducted. Clinical data of 1204 patients with gastric cancer who underwent laparoscopic radical gastrectomy at the First Affiliated Hospital of Army Medical University from April 2009 to December 2018 were collected. The digestive tract reconstruction methods included Billroth II anastomosis, Roux-en-Y anastomosis and un-cut-Roux- en-Y anastomosis. A linear stapler was used to transected the stomach and the duodenum. Among 1204 patients, 838 were males and 366 were females with mean age of (57.0±16.0) years. Duodenal stump was reinforced in 792 cases (reinforcement group) and unreinforced in 412 cases (non-reinforcement group). There were significant differences in resection range and anastomotic methods between the two groups (both P<0.001). The two groups were matched by propensity score according to the ratio of 1â¶1, and the reinforcement group was further divided into purse string group and non-purse string group. The primary outcome was short-term postoperative complications (within one month after operation). Complications with Clavien-Dindo grade ≥ III a were defined as severe complications, and the morbidity of complication between the reinforcement group and the non-reinforcement group, as well as between the purse string group and the non-purse string group was compared. Results: After PSM, 411 pairs were included in the reinforcement group and the non-reinforcement group, and there were no significant differences in baseline data between the two groups (all P>0.05). No perioperative death occurred in any patient.The short-term morbidity of postoperative complication was 7.4% (61/822), including 14 cases of anastomotic leakage (23.0%), 11 cases of abdominal hemorrhage (18.0%), 8 cases of duodenal stump leakage (13.1%), 2 cases of incision dehiscence (3.3%), 6 cases of incision infection (9.8%) and 20 cases of abdominal infection (32.8%). Short-term postoperative complications were found in 25 patients (6.1%) and 36 patients (8.8%) in the reinforcement group and the non-reinforcement group, respectively, without significant difference (χ2=2.142, P=0.143). Nineteen patients (2.3%) developed short-term severe complications (Clavien-Dindo grade ≥IIIa), while no significant difference in severe complications was found between the two groups (1.7% vs. 2.9%, χ2=1.347, P=0.246). Sub-group analysis showed that the morbidity of short-term postoperative complication of the purse string group was 2.6% (9/345), which was lower than 24.2% (16/66) of the non-purse string group (χ2=45.388, P<0.001). Conclusion: Conventional reinforcement of duodenal stump does not significantly reduce the incidence of duodenal stump leakage, so it is necessary to choose whether to reinforce the duodenal stump individually, and purse string suture should be the first choice when decided to reinforce.
Assuntos
Laparoscopia , Neoplasias Gástricas , Adulto , Idoso , Anastomose em-Y de Roux/efeitos adversos , Anastomose Cirúrgica/efeitos adversos , Duodeno/cirurgia , Feminino , Gastrectomia/métodos , Humanos , Laparoscopia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Estudos Retrospectivos , Neoplasias Gástricas/cirurgiaRESUMO
Objective: To compare the clinical efficacy and quality of life between uncut Roux-en-Y and Billroth II with Braun anastomosis in laparoscopic distal gastrectomy for gastric cancer patients. Methods: A retrospective cohort study was performed. Inclusion criteria: (1) 18 to 75 years old; (2) gastric cancer proved by preoperative gastroscopy, CT and pathological results and tumor was suitable for D2 radical distal gastrectomy; (3) postoperative pathological diagnosis stage was T1-4aN0-3M0 (according to the AJCC-7th TNM tumor stage), and the margin was negative; (4) Eastern Cooperative Oncology Group (ECOG) physical status score <2 points, and American Association of Anesthesiologists (ASA) grade 1 to 3; (5) no mental illness; (6) able to answer questionnaires independently; (7) patients agreed to undergo laparoscopic distal gastrectomy and signed an informed consent. Exclusion criteria: (1) patients with severe chronic diseases and American Association of Anesthesiologists (ASA) grade >3; (2) patients with other malignant tumors; (3) patients suffered from serious mental diseases; (4) patients received neoadjuvant chemotherapy or immunotherapy. According to the above criteria, clinical data of 200 patients who underwent laparoscopic distal gastrectomy at the Department of General Surgery of the First Affiliated Hospital of Army Medical University from January 2016 to December 2019 were collected. Of the 200 patients, 108 underwent uncut Roux-en-Y anastomosis and 92 underwent Billroth II with Braun anastomosis. The general data, intraoperative and postoperative conditions, complications, and endoscopic evaluation 1 year after the surgery were compared. Besides, the quality of life of two groups was also compared using the Chinese version of the European Organization For Research and Treatment of Cancer (EORTC) quality of life questionnaire-Core 30 (QLQ-C30) and quality of life questionnaire-stomach 22 (QLQ-STO22). Results: There were no significant differences in baseline data between the two groups (all P>0.05). All the 200 patients successfully underwent laparoscopic distal gastrectomy without intraoperative complications, conversion to open surgery or perioperative death. There were no significant differences between two groups in operative time, intraoperative blood loss, postoperative complications, time to flatus, time to removal of gastric tube, time to liquid diet, time to removal of drainage tube or length of postoperative hospital stay (all P>0.05). Endoscopic evaluation was conducted 1 year after surgery. Compared to Billroth II with Braun group, the uncut Roux-en-Y group had a significantly lower incidences of gastric stasis [19.8% (17/86) vs. 37.0% (27/73), χ(2)=11.199, P=0.024], gastritis [11.6% (10/86) vs. 34.2% (25/73), χ(2)=20.892, P<0.001] and bile reflux [1.2% (1/86) vs. 28.8% (21/73), χ(2)=25.237, P<0.001], and the differences were statistically significant. The EORTC questionnaire was performed 1 year after surgery, there were no significant differences in the scores of QLQ-C30 scale between the two groups (all P>0.05), while the scores of QLQ-STO22 showed that, compared to the Billroth II with Braun group, the uncut Roux-en-Y group had a lower pain score (median: 8.3 vs. 16.7, Z=-2.342, P=0.019) and reflux score (median: 0 vs 5.6, Z=-2.284, P=0.022), and the differences were statistically significant (all P<0.05), indicating milder symptoms. Conclusion: The uncut Roux-en-Y anastomosis is safe and reliable in laparoscopic distal gastrectomy, which can reduce the incidences of gastric stasis, gastritis and bile reflux, and improve the quality of life of patients after surgery.
Assuntos
Laparoscopia , Neoplasias Gástricas , Adolescente , Adulto , Idoso , Anastomose em-Y de Roux/efeitos adversos , Anastomose Cirúrgica/efeitos adversos , Gastrectomia/métodos , Gastroenterostomia/efeitos adversos , Humanos , Laparoscopia/métodos , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Qualidade de Vida , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Resultado do Tratamento , Adulto JovemRESUMO
Biliary atresia (BA) is a destructive bile duct disease occurring in newborn children within a few weeks after birth. In this study, the effect of miR-29c and miR-129-5p on epithelial-mesenchymal transition (EMT) in experimental BA was explored by constructing BA mouse models via Rhesus rotavirus vaccine infection. miR-29c and miR-129-5p expression was analyzed by real-time quantitative polymerase chain reaction. EMT was established by induction with transforming growth factor (TGF)-ß1. miR-29c and miR-129-5p were overexpressed and inhibited, respectively, by Lipofectamine transfection. EMT-related protein (formin-like 2, FMNL2; E-cadherin; vimentin; and cytokeratin-19, CK-19) expression was analyzed by western blot and immunofluorescent assay. The results indicated that miR-29c and miR-129-5p were downregulated and upregulated in BA mice. TGF-ß1 induction caused a time-dependent decrease and increase in miR-29c and miR-129-5p, respectively. Additionally, TGF-ß1 induced an increase in FMNL2 and vimentin expression and a decrease in E-cadherin and CK-19 expression (P < 0.05). Overexpression or suppression of miRNA-29c or miR-129-5p, respectively, induced the inhibition of FMNL2 and vimentin, and promotion of E-cadherin and CK-19 expression, in the test groups compared to the non-intervention group (P < 0.05). However, the FMNL2, vimentin, E-cadherin, and CK- 19 expression did not differ between the control and non-intervention groups (P > 0.05). Thus, miR-29c upregulation or miR-129-5p downregulation effectively prevented EMT in BA by regulating the expression of EMT pathway-related proteins. Therefore, miR-29c and miR-129-5p could be utilized as therapeutic targets for BA in the future.
Assuntos
Atresia Biliar/genética , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MicroRNAs/genética , Fator de Crescimento Transformador beta1/farmacologia , Animais , Animais Recém-Nascidos , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Atresia Biliar/etiologia , Atresia Biliar/metabolismo , Atresia Biliar/patologia , Caderinas/genética , Caderinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Forminas , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratina-19/genética , Queratina-19/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Cultura Primária de Células , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/efeitos adversos , Transdução de Sinais , Vimentina/genética , Vimentina/metabolismoRESUMO
Sphingolipids are major components of the plasma membrane of eukaryotic cells and were once thought of merely as structural components of the membrane. We have investigated effects of inhibiting sphingolipid biosynthesis, both in germinating spores and growing hyphae of Aspergillus nidulans. In germinating spores, genetic or pharmacological inactivation of inositol phosphorylceramide (IPC) synthase arrests the cell cycle in G(1) and also prevents polarized growth during spore germination. However, inactivation of IPC synthase not only eliminates sphingolipid biosynthesis but also leads to a marked accumulation of ceramide, its upstream intermediate. We therefore inactivated serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway, to determine effects of inhibiting sphingolipid biosynthesis without an accumulation of ceramide. This inactivation also prevented polarized growth but did not affect nuclear division of germinating spores. To see if sphingolipid biosynthesis is required to maintain polarized growth, and not just to establish polarity, we inhibited sphingolipid biosynthesis in cells in which polarity was already established. This inhibition rapidly abolished normal cell polarity and promoted cell tip branching, which normally never occurs. Cell tip branching was closely associated with dramatic changes in the normally highly polarized actin cytoskeleton and found to be dependent on actin function. The results indicate that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that accumulation of ceramide is likely responsible for arresting the cell cycle in G(1).
Assuntos
Aspergillus nidulans/metabolismo , Esfingolipídeos/biossíntese , Actinas/metabolismo , Aspergillus nidulans/citologia , Ciclo Celular , Polaridade Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestruturaRESUMO
The assembly of an oligosaccharide library has been achieved in a practical and efficient manner employing a' one-pot sequential approach. With the help of the anomeric reactivity values of thioglycosides, using a thioglycoside (mono- or disaccharide) with one free hydroxyl group as acceptor and donor coupled with another fully protected thioglycoside, a di- or trisaccharide is selectively formed without self-condensation and subsequently reacted in situ with an anomerically inactive glycoside (mono- or disaccharide) to form a tri- or tetrasaccharide in high overall yield. The approach enables the rapid assembly of 33 linear or branched fully protected oligosaccharides using designed building blocks. These fully protected oligosaccharides have been partially or completely deprotected to create 29 more structures to further increase the diversity of the library.
Assuntos
Oligossacarídeos/síntese química , Sequência de Carboidratos , Catálise , Radical Hidroxila , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligossacarídeos/química , Tioglicosídeos/químicaRESUMO
In Aspergillus nidulans, mutation of the transcriptional regulator brlA arrests formation of asexual spore-forming structures called conidiophores but does not hinder vegetative hyphal growth. During conidiophore development a 6-fold, brlA-dependent increase in the kinase activities of NIMX(cdc2) and NIMA occurs. A similar level of kinase induction was promoted by ectopic expression of brlA. Northern and Western analysis revealed marked induction of nimX(cdc2) mRNA after ectopic expression of brlA and increased amounts of NIMX(cdc2). Therefore, nimX(cdc2) is developmentally regulated by brlA indicating a direct role for brlA in the regulation of cell cycle genes. That correct regulation of nimX(cdc2) is important for normal development was further supported by analysis of conidiophore development and septation in cell cycle specific mutants. Most noticeably, the nimX(cdc2AF) mutation promoted inappropriate septation and hindered the switch from filamentous growth to budding growth seen during conidiophore development. Therefore, in contrast to the situation previously reported for other multicellular eukaryotes, interaction between developmental regulators and cell cycle regulators is essential for normal morphogenesis in A.nidulans.
Assuntos
Aspergillus nidulans/citologia , Aspergillus nidulans/fisiologia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Ciclinas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição , Aspergillus nidulans/genética , Divisão Celular , Ciclinas/metabolismo , Cinética , Morfogênese , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esporos Fúngicos/ultraestrutura , Dedos de ZincoRESUMO
The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMX(cdc2) in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsB(rad3) and uvsD(rad26) have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIME(cyclinB), but ectopic expression of active nondegradable NIME(cyclinB) does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMX(cdc2) and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.
Assuntos
Aspergillus nidulans/genética , Proteínas de Ciclo Celular , Replicação do DNA/genética , Mitose/genética , Proteínas de Schizosaccharomyces pombe , Adenosina Trifosfatases/genética , Proteína Quinase CDC2/genética , Dano ao DNA/genética , DNA Helicases/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Hidroxiureia/farmacologia , Mutação , Fosforilação , Fase S/genética , Proteínas de Saccharomyces cerevisiae , Raios UltravioletaRESUMO
Surprisingly, although highly temperature-sensitive, the bimA1(APC3) anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1(APC3) is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34(cdc2) kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1(APC3)-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1(APC3) double mutant arrests in a mitotic state with very high p34(cdc2) H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1(APC3) mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1(APC3). The bimA1(APC3) mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.
Assuntos
Aspergillus nidulans/metabolismo , Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Ligases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Complexos Ubiquitina-Proteína Ligase , Anáfase , Ciclossomo-Complexo Promotor de Anáfase , Aspergillus nidulans/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Ciclinas/metabolismo , Endopeptidases/metabolismo , Proteínas Fúngicas/genética , Fase G2 , Mitose , Mutagênese , Mutação , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína LigasesRESUMO
Prevention of mitosis if DNA is damaged, or not fully replicated, is a widespread mechanism used by eukaryotic cells to maintain their ploidy and prevent accumulation of mutations. Such 'checkpoints' must inhibit mitotic regulators to prevent mitotic progression when DNA is not ready for segregation. The mitotic regulators targeted for negative regulation by these checkpoints differ among cell types, but two conserved targets have emerged, the anaphase-promoting complex (APC) and tyrosine phosphorylation of p34(cdc2). One potential downstream target of both these regulators has also been identified, the mitosis-promoting NIMA kinase.
RESUMO
It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.
Assuntos
Aspergillus nidulans/citologia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular , Dano ao DNA , Mitose/genética , Tirosina/metabolismo , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/efeitos da radiação , Proteína Quinase CDC2/antagonistas & inibidores , Reparo do DNA , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , Fase G2/genética , Metanossulfonato de Metila/farmacologia , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Mutagênicos/farmacologia , Mutação , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Raios UltravioletaAssuntos
Aspergillus nidulans/enzimologia , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Anticorpos , Aspergillus nidulans/citologia , Aspergillus nidulans/crescimento & desenvolvimento , Sítios de Ligação , Cromatografia de Afinidade , Estabilidade Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Cinética , Dados de Sequência Molecular , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/isolamento & purificação , Especificidade por Substrato , TermodinâmicaRESUMO
In A. nidulans, activation of both p34cdc2/cyclinB H1 and NIMA kinases is required to initiate mitosis. These two kinases are regulated at several levels during interphase and are activated independently as protein kinases during G2. They are also targeted for negative regulation, to prevent mitosis by mitotic entry checkpoint controls, when DNA is not replicated or is damaged. Then, to initiate mitosis, they promote each other's mitotic functions to coordinately promote mitosis upon completion of interphase events. In addition, inactivation of both kinases by mitotic specific proteolysis is also required for progression through mitosis into G1.
Assuntos
Aspergillus nidulans/citologia , Aspergillus nidulans/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular , Ciclina B/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Dano ao DNA , Inibidores Enzimáticos/metabolismo , Fase G2/fisiologia , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Proteínas Serina-Treonina Quinases/antagonistas & inibidoresRESUMO
Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Replicação do DNA , Proteínas Fúngicas/metabolismo , Tirosina/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Aspergillus , Ciclo Celular , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Mitose , Dados de Sequência Molecular , FosforilaçãoRESUMO
Great progress has recently been made in our understanding of the regulation of the eukaryotic cell cycle, and the central role of cyclin-dependent kinases is now clear. In Aspergillus nidulans it has been established that a second class of cell-cycle-regulated protein kinases, typified by NIMA (encoded by the nimA gene), is also required for cell cycle progression into mitosis. Indeed, both p34cdc2/cyclin B and NIMA have to be correctly activated before mitosis can be initiated in this species, and p34cdc2/cyclin B plays a role in the mitosis-specific activation of NIMA. In addition, both kinases have to be proteolytically destroyed before mitosis can be completed. NIMA-related kinases may also regulate the cell cycle in other eukaryotes, as expression of NIMA can promote mitotic events in yeast, frog or human cells. Moreover, dominant-negative versions of NIMA can adversely affect the progression of human cells into mitosis, as they do in A. nidulans. The ability of NIMA to influence mitotic regulation in human and frog cells strongly suggests the existence of a NIMA pathway of mitotic regulation in higher eukaryotes. A growing number of NIMA-related kinases have been isolated from organisms ranging from fungi to humans, and some of these kinases are also cell-cycle-regulated. How NIMA-related kinases and cyclin-dependent kinases act in concert to promote cell cycle transitions is just beginning to be understood. This understanding is the key to a full knowledge of cell cycle regulation.
Assuntos
Aspergillus nidulans/citologia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular , Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Aspergillus nidulans/enzimologia , Humanos , Mitose , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , FosforilaçãoRESUMO
We demonstrate that there are at least two S-phase checkpoint mechanisms controlling mitosis in Aspergillus. The first responds to the rate of DNA replication and inhibits mitosis via tyrosine phosphorylation of p34cdc2. Cells unable to tyrosine phosphorylate p34cdc2 are therefore viable but are unable to tolerate low levels of hydroxyurea and prematurely enter lethal mitosis when S-phase is slowed. However, if the NIMA mitosis-promoting kinase is inactivated then non-tyrosine-phosphorylated p34cdc2 cannot promote cells prematurely into mitosis. Lack of tyrosine-phosphorylated p34cdc2 also cannot promote mitosis, or lethality, if DNA replication is arrested, demonstrating the presence of a second S-phase checkpoint mechanism over mitotic initiation which we show involves the function of BIME. In order to overcome the S-phase arrest checkpoint over mitosis it is necessary both to prevent tyrosine phosphorylation of p34cdc2 and also to inactivate BIME. Lack of tyrosine phosphorylation of p34cdc2 allows precocious expression of NIMA during S-phase arrest, and lack of BIME then allows activation of this prematurely expressed NIMA by phosphorylation. The mitosis-promoting NIMA kinase is thus a target for S-phase checkpoint controls.
Assuntos
Aspergillus nidulans/citologia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular , Proteínas Fúngicas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Tirosina/metabolismo , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Sequência de Bases , Proteína Quinase CDC2/antagonistas & inibidores , DNA/biossíntese , DNA/efeitos dos fármacos , Primers do DNA , Hidroxiureia/farmacologia , Mitose , Dados de Sequência Molecular , Mutação , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de SinaisRESUMO
To investigate the degree of conservation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans, and to help direct its functional analysis, we cloned a homolog (designated nim-1) from Neurospora crassa. Over the catalytic domain NIM-1 is 75% identical to NIMA, but overall the identity drops to 52%. nim-1 was able to functionally complement nimA5 in A. nidulans. Mutational analysis of potential activating phosphorylation sites found in NIMA, NIM-1, and related protein kinases was performed on NIMA. Mutation of threonine 199 (conserved in all NIMA-related kinases) inhibited NIMA beta-casein kinase activity and abolished its in vivo function. This site conforms to a minimal consensus phosphorylation site for NIMA (FXXT) and is analogous to the autophosphorylation site of cyclic-AMP-dependent protein kinases. However, mutation of a unique cysteine residue found only in the catalytic site of NIMA and NIM-1 had no effect on NIMA kinase activity or function. Three temperature-sensitive alleles of nimA that cause arrest in G2 were sequenced and shown to generate three different amino acid substitutions. None of the mutations prevented accumulation of NIMA protein during G2 arrest, but all prevented the p34cdc2/cyclin B-dependent phosphorylation of NIMA normally seen during mitotic initiation even though p34cdc2/cyclin B H1 kinase activity was fully activated.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas de Ciclo Celular , Ciclo Celular , Sequência Conservada , Proteínas Serina-Treonina Quinases/isolamento & purificação , Sequência de Aminoácidos , Aspergillus nidulans/citologia , Sequência de Bases , Catálise , Clonagem Molecular , Cisteína/genética , Análise Mutacional de DNA , DNA Fúngico , Ativação Enzimática , Mitose , Dados de Sequência Molecular , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Neurospora crassa/genética , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34cdc2/cyclin B and NIMA. Forced expression of NIMA, even when p34cdc2 was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitosis-promoting function of NIMA is normally under control of p34cdc2/cyclin B as the active G2 form of NIMA is hyperphosphorylated and further activated by p34cdc2/cyclin B when cells initiate mitosis. To see the p34cdc2/cyclin B dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34cdc2/cyclin B generated the MPM-2 epitope, suggesting that NIMA is phosphorylated directly by p34cdc2/cyclin B during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest that each activates the other's mitosis-promoting functions. This ensures that cells coordinately activate p34cdc2/cyclin B and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA is regulated through the cell cycle like cyclin B, as it accumulates during G2 and is degraded only when cells traverse mitosis.
Assuntos
Aspergillus nidulans/enzimologia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular , Ciclinas/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Aspergillus nidulans/citologia , Sequência de Bases , Cromatina/fisiologia , Éteres Cíclicos/farmacologia , Fase G2 , Modelos Biológicos , Dados de Sequência Molecular , Mutação/fisiologia , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas/biossíntese , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologiaRESUMO
After the merozoite entered the erythrocyte, the membrane debris in the parasitophorous vacuoles of early ring form was passed out through a narrow external aperture in erythrocyte to the exterior. The trophozoite was oval or irregular in shape. Ingestion of host cell cytoplasm occurred cystostomally. The asexual parasite possessed cristate mitochondria and was surrounded by a single-membraned pellicle. The gametocyte possessed cristate mitochondria and was surrounded by two unit membranes. The cytoplasm of mature macrogametocytes contained many ribosomes, mitochondria and osmiophilic bodies and a small nucleus while microgametocytes contained fewer ribosomes, osmiophilic bodies and mitochondria and a large nucleus. Three characteristic morphological alterations were observed within the host cells, that is, small vesicles, cytoplasmic cleft and caveola-vesicle complex. The clefts within the cytoplasm of the host erythrocytes were present in all human malarial parasites. The small vesicles distributed all over the cytoplasm were surrounded by a unit membrane. The caveola-vesicle complex consisted of caveolae was surrounded by small vesicles and probably corresponds to a Schüffner's dot. (Figs. 1-13).