RESUMO
BACKGROUND: Perineal hernia (PH) is a late complication of abdominoperineal resection (APR) that may compromise a patient's quality of life. The frequency and risk factors for PH after robotic APR adopting recent rectal cancer treatment strategies remain unclear. METHODS: Patients who underwent robotic APR for rectal cancer between December 2011 and June 2022 were retrospectively examined. From July 2020, pelvic reinforcement procedures, such as robotic closure of the pelvic peritoneum and levator ani muscles, were performed as prophylactic procedures for PH whenever feasible. PH was diagnosed in patients with or without symptoms using computed tomography 1 year after surgery. We examined the frequency of PH, compared characteristics between patients with PH (PH+) and without PH (PH-), and identified risk factors for PH. RESULTS: We evaluated 142 patients, including 53 PH+ (37.3%) and 89 PH- (62.6%). PH+ had a significantly higher rate of preoperative chemoradiotherapy (26.4% versus 10.1%, p = 0.017) and a significantly lower rate of undergoing pelvic reinforcement procedures (1.9% versus 14.0%, p = 0.017). PH+ had a lower rate of lateral lymph node dissection (47.2% versus 61.8%, p = 0.115) and a shorter operative time (340 min versus 394 min, p = 0.110). According to multivariate analysis, the independent risk factors for PH were preoperative chemoradiotherapy, not undergoing lateral lymph node dissection, and not undergoing a pelvic reinforcement procedure. CONCLUSIONS: PH after robotic APR for rectal cancer is not a rare complication under the recent treatment strategies for rectal cancer, and performing prophylactic procedures for PH should be considered.
Assuntos
Períneo , Complicações Pós-Operatórias , Protectomia , Neoplasias Retais , Procedimentos Cirúrgicos Robóticos , Humanos , Estudos Retrospectivos , Procedimentos Cirúrgicos Robóticos/efeitos adversos , Procedimentos Cirúrgicos Robóticos/métodos , Masculino , Feminino , Fatores de Risco , Pessoa de Meia-Idade , Períneo/cirurgia , Idoso , Protectomia/efeitos adversos , Protectomia/métodos , Neoplasias Retais/cirurgia , Incidência , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/epidemiologia , Hérnia/etiologia , Hérnia/prevenção & controle , Hérnia/epidemiologia , Hérnia Incisional/etiologia , Hérnia Incisional/prevenção & controle , Hérnia Incisional/epidemiologiaRESUMO
PURPOSE: Colostomy is a common procedure for fecal diversion, but the optimal colostomy approach is unclear in terms of surgical outcomes and stoma-related complications. The purpose of this study was to examine the efficacy and feasibility of laparoscopic loop colostomy. METHODS: This retrospective cohort study included patients who underwent loop colostomy at Shizuoka Cancer Center in Japan between April 2010 and March 2022. Patients were divided into two groups based on surgical approach: the laparoscopic (LAP) and open (OPEN) groups. Surgical outcomes and the incidences of stoma-related complications such as stomal prolapse (SP), parastomal hernia (PSH), and skin disorders (SD) were compared with and without propensity score matching. RESULTS: Of the 388 eligible patients, 180 (46%) were in the LAP group and 208 (54%) were in the OPEN group. The male-to-female ratio was 5.5:4.5 in the Lap group and was 5.3:4.7 in the OPEN group, respectively. The median age was 68 years (range, 31-88 years) in the LAP group and 65 years (range, 23-93 years) in the OPEN group, respectively. The LAP group, compared with the OPEN group, had a shorter operative time and lower incidences of surgical site infection (3.9% versus 16.3%, respectively; p < 0.01) and SD (11.7% versus 24.5%, respectively; p < 0.01). There was no significant difference between the LAP and OPEN groups in the incidence of SP (17.3% versus 17.3%, respectively) or PSH (8.9% versus 6.7%, respectively). After propensity score matching, the incidences of surgical site infection and SD were significantly lower in the LAP group than in the OPEN group, while there were no significant differences in the operative time or the incidences of SP and PSH. CONCLUSION: Our results suggest that laparoscopic surgery could be beneficial and feasible in loop colostomy.
Assuntos
Hérnia Incisional , Laparoscopia , Humanos , Masculino , Feminino , Idoso , Colostomia/efeitos adversos , Colostomia/métodos , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/etiologia , Estudos Retrospectivos , Pontuação de Propensão , Laparoscopia/efeitos adversos , Laparoscopia/métodosRESUMO
BACKGROUND: What qualifies as optimal lymph node (LN) dissection in the surgical management of splenic flexure colon cancer (SFCC) still remains controversial because few studies have evaluated the distribution of LN metastasis of SFCC. The aim of this study was to clarify detailed distribution of LN metastasis and long-term outcomes of SFCC. METHODS: This retrospective study enrolled patients who had curative colectomy for primary transverse or descending colon cancer of pathological stage I, II, or III at a single high-volume cancer center between April 2002 and December 2018. The 538 eligible patients were divided into three groups: patients with SFCC (SFCC group, n = 168), patients with proximal transverse colon cancer (PTCC group, n = 290), and patients with distal descending colon cancer (DDCC group, n = 80). LNs were classified into horizontal (pericolic) and vertical (intermediate and main) nodes. Intermediate and main LN station numbers were defined according to the Japanese Society for Cancer of the Colon and Rectum classification. Distributions of LN metastasis and long-term outcomes were compared. RESULTS: In the SFCC group, the mean age was 67.3 ± 10.5 years and 110 patients (65.5%) were male. The proportion of patients with LN metastasis in the intermediate or main region was significantly lower in the SFCC group (8%) than in the PTCC (37%) (p < 0.01) or DDCC group (29%) (p < 0.01) in pathological stage III patients. In the SFCC group, the incidence of pericolic LN metastasis on the oral side of tumor (43%) was significantly higher than in the PTCC group (21%) (p < 0.01) and was similar to that in the DDCC group (42%) (p = 0.51), while in the SFCC group, the incidence of pericolic LN metastasis on the anal side of tumor (17%) was lower than in the PTCC group (31%) and was also similar to that in the DDCC group (21%). There were no significant differences in disease-specific survival rates among all groups. CONCLUSIONS: LN metastasis occurred mainly in the pericolic region, especially on the oral side of the tumor in SFCC. It may, therefore, be important to have an adequate bowel resection margin, especially on the oral side, for SFCC.
Assuntos
Colo Transverso , Neoplasias do Colo , Idoso , Colo Transverso/cirurgia , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Feminino , Humanos , Excisão de Linfonodo , Linfonodos/patologia , Linfonodos/cirurgia , Masculino , Margens de Excisão , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos RetrospectivosRESUMO
BACKGROUND: The safety and feasibility of robotic-assisted multivisceral resection for locally advanced rectal cancer remain unclear. The aim of this study was to assess the short-term outcomes of this procedure at our institution. METHODS: From December 2011 to December 2016, patients who underwent robotic-assisted multivisceral resection for rectal cancer were investigated. Patient demographics, treatment characteristics, perioperative outcomes, and pathological results were evaluated retrospectively. RESULTS: There were 31 patients; 17 men (54.8%) and 14 women (45.2%), with a median age of 65 years (range 40-82 years). Twenty-one patients (67.7%) had a cT4 tumor, 9 patients (29.0%) had a pT4b tumor, and all patients except one (96.8%) underwent complete resection of the primary tumor with negative resection margins. Eleven patients (35.5%) received neoadjuvant chemoradiation. The most commonly resected organ was the vaginal wall (n = 12, 38.7%), followed by the prostate (n = 10, 32.3%). Lateral lymph node dissection was performed in 20 patients (64.5%). The median operative time was 394 min (range 189-549 min), and the median blood loss was 41 mL (range 0-502 mL). None of the patients received intraoperative blood transfusions or required conversion to open. Overall, postoperative complications occurred in 11 patients (35.5%). The most frequent complication was urinary retention (n = 5, 16.1%), and none of the patients developed serious complications classified as Clavien-Dindo grades III-V. CONCLUSIONS: Robotic-assisted multivisceral resection for rectal cancer is safe and technically feasible.
Assuntos
Genitália Masculina/cirurgia , Neoplasias Retais/cirurgia , Procedimentos Cirúrgicos Robóticos/métodos , Vagina/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Perda Sanguínea Cirúrgica , Feminino , Genitália Masculina/patologia , Humanos , Excisão de Linfonodo , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Duração da Cirurgia , Complicações Pós-Operatórias/etiologia , Próstata/patologia , Próstata/cirurgia , Neoplasias Retais/patologia , Estudos Retrospectivos , Procedimentos Cirúrgicos Robóticos/efeitos adversos , Glândulas Seminais/patologia , Glândulas Seminais/cirurgia , Fatores de Tempo , Resultado do Tratamento , Bexiga Urinária/patologia , Bexiga Urinária/cirurgia , Vagina/patologia , Ducto Deferente/patologia , Ducto Deferente/cirurgiaRESUMO
Reduction in eel resources and catches of glass eels as seedlings for aquaculture have been a serious concern in recent years in both Europe and East Asia. Thus, technical advancement to produce eel seeds for artificial cultivation is most desired. Fundamental information on oocyte maturation and ovulation and its application to artificial induction of sexual maturation are needed to produce good quality seeds of the Japanese eel. This review introduces hormonal mechanisms of cytoplasmic maturation (such as hydration, lipid coalescence, and clearing of the ooplasm) and the maturational competence (the ability to respond to maturation-inducing steroid) and nuclear maturation (germinal vesicle breakdown). In addition, previous and newly developed methods for induction of spawning have been described.
Assuntos
Aquicultura/métodos , Cruzamento/métodos , Citoplasma/fisiologia , Enguias/fisiologia , Oócitos/crescimento & desenvolvimento , Ovulação/fisiologia , Maturidade Sexual/fisiologia , Animais , Feminino , Japão , Modelos Biológicos , Técnicas de Reprodução Assistida/veterináriaRESUMO
Tryptophan hydroxylase (tph) is the key regulator in serotonin (5-HT) biosynthesis that stimulates the release of GnRH and gonadotropins by acting at the level of hypothalamo-hypophyseal axis. In brain, 5-HT is expressed predominantly in preoptic area-hypothalamus (POA-HYP) region in teleosts. Therefore, in the present study we isolated tph2 from catfish brain to evaluate its expression pattern in male and female brains during early development. Tph2 cloned from catfish brain is 2.768 Kb in length which encodes predicted protein of 488 amino acid residues. The characterization of recombinant tph2 was done by transient transfection in CHO cells. Tissue distribution of tph2 revealed ubiquitous expression except ovary. Real time PCR analysis in discrete regions of adult male brain revealed that tph2 mRNA was abundant in the POA-HYP and optic tectum+cerebellum+thalamus (OCT) regions. Differential expression of tph2 was observed at mRNA and protein levels in the POA-HYP and OCT regions of male and female brains during development that further correlate with the 5-hydroxytryptophan (5-HTP) and 5-HT levels measured using HPLC method in these regions of male and female brains. Tph2 immunoreactive neurons were observed in different regions of brain at 50 days post hatch using catfish specific tph2 antibody. Changes in tph2 mRNA expression, 5-HTP, and 5-HT levels in the POA-HYP+OCT region of brains of methyltestosterone and para-chlorophenylalanine treated fishes during development further endorse our results. Based on our results, we propose that the serotonergic system is involved in brain sex differentiation in teleosts.
Assuntos
5-Hidroxitriptofano/metabolismo , Encéfalo/metabolismo , Serotonina/metabolismo , Diferenciação Sexual/fisiologia , Triptofano Hidroxilase/genética , Animais , Western Blotting , Encéfalo/enzimologia , Células CHO , Peixes-Gato , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Feminino , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Diferenciação Sexual/genéticaRESUMO
Serotonin (5-HT) is well known for modulating the release of GnRH and gonadotropin in teleosts. Reports on increased female:male ratio after the blockade of 5-HT biosynthesis proposed a role for 5-HT in brain sex differentiation. Two types of tryptophan hydroxylase (Tph), rate-limiting enzyme in the biosynthesis of 5-HT were cloned from vertebrates. In the present study, we cloned Tph from brain and evaluated its importance during early development of XX and XY Nile tilapia. Tph cloned from tilapia brain is 1888 bp in length and it encodes predicted protein of 462 amino acid residues. Tph activity of tilapia was confirmed by demonstrating the conversion of L-tryptophan to 5-hydroxy tryptophan by the recombinant protein after transient transfection of this cDNA clone in COS-7 cells. Northern blot identified single transcript around 2kb in male brain. Tissue distribution of Tph revealed high abundance in brain, kidney, liver and testis. Semi-quantitative RT-PCR revealed exclusive expression of Tph in the male brain from 5 to 20 days post hatch (dph) while in the female brain, it was from 25 dph. These results were authenticated by localization of Tph transcripts in olfactory bulb-telencephalon region of 11 dph male brain using in situ hybridization. Tph immunoreactivity (-ir) was also evident in the nucleus preopticus-periventricularis area of male brain as early as 12 dph. However, Tph-ir was observed in several regions of both male and female brain without any distinction from 30 dph. Dimorphic expression pattern of Tph during early brain development around the critical period (7-21 dph) of gonadal sex determination and differentiation may implicate a role for Tph in brain sex differentiation of tilapia.
Assuntos
Encéfalo/enzimologia , Expressão Gênica , Caracteres Sexuais , Tilápia/crescimento & desenvolvimento , Tilápia/metabolismo , Triptofano Hidroxilase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células COS , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Bulbo Olfatório/enzimologia , RNA Mensageiro/análise , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Telencéfalo/enzimologia , Triptofano Hidroxilase/análise , Triptofano Hidroxilase/químicaRESUMO
We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Expressão Gênica , Genes de Helmintos , Troponina C/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Clonagem Molecular , Drosophila melanogaster/genética , Imunofluorescência , Dados de Sequência Molecular , Desenvolvimento Muscular , Músculos/embriologia , Músculos/metabolismo , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Troponina C/química , Troponina C/metabolismo , Troponina I/metabolismoRESUMO
Traveling, living and working in space is now a reality. The number of people and length of time in space is increasing. With new horizons for exploration it becomes more important to fully understand and provide countermeasures to the effects of the space environment on the human body. In addition, space provides a unique laboratory to study how life and physiologic functions adapt from the cellular level to that of the entire organism. Caenorhabditis elegans is a genetic model organism used to study physiology on Earth. Here we provide a description of the rationale, design, methods, and space culture validation of the ICE-FIRST payload, which engaged C. elegans researchers from four nations. Here we also show C. elegans growth and development proceeds essentially normally in a chemically defined liquid medium on board the International Space Station (10.9 day round trip). By setting flight constraints first and bringing together established C. elegans researchers second, we were able to use minimal stowage space to successfully return a total of 53 independent samples, each containing more than a hundred individual animals, to investigators within one year of experiment concept. We believe that in the future, bringing together individuals with knowledge of flight experiment operations, flight hardware, space biology, and genetic model organisms should yield similarly successful payloads.
RESUMO
BACKGROUND/AIMS: The age-associated dysregulation of hemodynamic, metabolic and immune responses contributes to the high incidence of complications after major abdominal surgery. METHODOLOGY: Ninety-five patients who underwent gastric resection (n=51) and colorectal resection (n=44) were divided according to age into Groups A (n=45, less than 70 years old), B (n=30, 70-79 years) and C (n=20, over 80 years). Flow cytometric analysis of CD4+ lymphocytes for interferon (IFN)-gamma and interleukin (IL)-4 production determined the Th1/2 balance. Energy expenditure was measured by indirect calorimetry, and hemodynamics were studied using pulse dye densitometry. RESULTS: Surgical procedures, operating time, blood loss and morbidity did not significantly differ among the three groups. The cardiac index (CI) in group A and B increased significantly over preoperative levels until POD 3, but there were no significant perioperative changes in the CI levels of group C. Resting energy expenditure levels changed similarly to those of CI. The postoperative Th1/2 ratio decreased from young to elderly to very elderly patients, although no differences were significant before surgery. The postoperative percentage of CD4+IFN-gamma +T cells (Th1) in group C decreased significantly despite of no significant changes in that of group A and B. In contrast, the ratio of CD4+IL-4+T cells (Th2) in the all groups significantly increased after surgery. CONCLUSIONS: Host responses in elderly patients after major abdominal surgery were more hyperdynamic and hypermetabolic than those of young patients. Postoperative dysregulation of the Th1/2 balance was also associated with aging. However, host responses appear to significantly differ between elderly and very elderly patients.
Assuntos
Colectomia , Gastrectomia , Idoso , Idoso de 80 Anos ou mais , Perda Sanguínea Cirúrgica , Volume Sanguíneo , Calorimetria Indireta , Débito Cardíaco , Metabolismo Energético , Feminino , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-4/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Células Th1 , Células Th2RESUMO
Polyhooks were isolated from Salmonella SJW880, a non-flagellated mutant, and purified by cesium chloride density gradient centrifugation. The polyhooks were disintegrated into protein subunits (monomer) by heat in the absence of salt. The monomer was repolymerized in the presence of moderately high concentrations of sodium citrate at neutral pH. Three types of polymer were produced. One type of polymer, produced at room temperature and at citrate concentrations less than 0.3 M, had no regular shape and no definite thickness. Another type of polymer, produced at room temperature and at citrate concentrations greater than 0.4 M, had a straight shape and a similar thickness to that of polyhook but was easily dissociated into monomer in the absence of salt. A third type of polymer was produced at low temperature, independently of the concentration of citrate, and seemed to be a tubular polymer with a thickness similar to that of polyhook but had no helical curvature. However, this type of polymer was shown to have a structure locally the same as that of polyhook by electron microscopic observation, optical diffraction and circular dichroism measurements.
Assuntos
Proteínas de Bactérias , Flagelos/análise , Salmonella/análise , Proteínas de Bactérias/genética , Dicroísmo Circular , Citratos , Microscopia Eletrônica , Mutação , Polímeros , Conformação Proteica , Salmonella/genéticaRESUMO
We have obtained 2-dimensional crystals of the beta-subunits of the chaperonin TF55 from Sulfolobus shibatae reconstituted into oligomers in the absence of alpha-subunits. The subunits form rings with 9-fold rotational symmetry which arrange themselves in a trigonal lattice. From electron micrographs of negatively stained specimens we have calculated a projection map in plane group p312 showing the rings in top-view.
Assuntos
Proteínas de Choque Térmico/isolamento & purificação , Chaperonas Moleculares/isolamento & purificação , Sulfolobus/metabolismo , Proteínas Arqueais , Cristalização , Proteínas de Choque Térmico/química , Microscopia Eletrônica , Chaperonas Moleculares/química , Mutagênese Sítio-DirigidaRESUMO
Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.
Assuntos
Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Troponina C/metabolismo , Aminoquinolinas , Animais , Sítios de Ligação , Evolução Molecular , Corantes Fluorescentes , Cinética , Mutação , Espectrometria de Fluorescência , Titulometria , Troponina C/genéticaRESUMO
To characterize excitation-contraction coupling in Caenorhabditis elegans, we applied two approaches. First, we isolated a mutant having abnormal responses to ketamine, an anesthetic in vertebrates. The novel mutation unc-68(kh30) (isolated as kra-1(kh30)), exhibited strict ketamine-dependent convulsions followed by paralysis. Second, we cloned the C. elegans ryanodine receptor gene ryr-1 that is located near the center of chromosome V. ryr-1 consists of 46 exons, which encode a predicted protein of 5071 amino acid residues that is homologous to Drosophila and vertebrate ryanodine receptors. ryr-1 promoter/lacZ plasmids were expressed in body-wall and pharyngeal muscles. Non-muscle cell expression may be seen with a truncated promoter. In addition, we show that the unc-68/kra-1(kh30) mutation is a Ser1444 Asn substitution at a putative protein kinase C phosphorylation site in ryr-1, and that unc-68(e540) contains a splice acceptor mutation that creates a premature stop codon in the ryr-1 gene. We confirmed that unc-68(e540) is a mutation in ryr-1 by injecting the complete ryr-1 gene into unc-68(e540) animals and recovering wild-type progeny. Results presented here will be useful in studying the structure and function of ryanodine receptors in excitation-contraction coupling and in understanding the evolution of ryanodine receptor tissue specificity.
Assuntos
Anestésicos Dissociativos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Canais de Cálcio/genética , Genes de Helmintos/genética , Ketamina/farmacologia , Proteínas Musculares/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Ácido Aspártico/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Canais de Cálcio/análise , Canais de Cálcio/metabolismo , Clonagem Molecular , Expressão Gênica , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Mutação , N-Metilaspartato/farmacologia , Especificidade de Órgãos , Fosforilação , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Proteínas Recombinantes de Fusão/análise , Canal de Liberação de Cálcio do Receptor de Rianodina , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Charge interactions between alpha-helical coiled-coil proteins have been postulated to determine the alignment of many filamentous proteins, such as myosin heavy-chain rod, paramyosin and alpha-keratin. Here we determined the sequence changes in nine mutations in the unc-15 paramyosin gene of Caenorhabditis elegans, including one nonsense, four missense, one deletion and three suppressor mutations. These mutation sites were located on a molecular model, constructed by optimizing charge interactions between paramyosin rods. Remarkably, single charge reversals (e.g., glutamic acid to lysine) were found that either disrupted or restored filament assembly in vivo. The positions of the mutations within the paramyosin molecule support the models of paramyosin assembly and further suggest that the C-terminal region containing a cluster of five mutations, and a site interacting with it, play a key role in assembly. One amino acid substitution in this C-terminal region, in which there is a "weak spot", led to a loss of reactivity with one monoclonal anti-paramyosin antibody. The results demonstrate how a single amino acid substitution can alter the assembly properties of alpha-helical molecules.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Caenorhabditis/genética , Músculos/fisiologia , Mutagênese Sítio-Dirigida , Tropomiosina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Músculos/ultraestrutura , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Conformação Proteica , Tropomiosina/fisiologia , Tropomiosina/ultraestruturaRESUMO
The tropomyosin gene tmy-1/lev-11 of Caenorhabditis elegans spans 14.5 kb and encodes three isoforms by alternative splicing. To identify, characterize and compare the genome and tissue expression of a fourth isoform, the technique of rapid amplification of cDNA ends and microinjection with lacZ and gfp fusion plasmids were employed. We elucidated CeTMIV, a fourth isoform of tmy-1, which encoded a 256 residue polypeptide. CeTMIV isoform had a similar promoter region to CeTMIII isoform, but was alternatively spliced to generate a cDNA that differed in two exons. The tmy-1::lacZ and tmy-1::gfp fusion genes, with 3.2 kb promoter sequence and 1.1 kb of CeTMIV isoform specific exons, were expressed in the pharyngeal and intestinal cells. Further unidirectional deletion of the sequence located the primary promoter region 853 bp upstream from the initial codon. We show within the upstream region, the presence of B and C subelement-like sequences of myo-2, which may be used to stimulate pharyngeal expression. Despite the presence of a ges-1 like sequence, we were unable to locate the two GATA sites required for intestinal expression. Reassessing tissue expression for CeTMIII isoform with newly constructed fusion plasmids, we showed further expression in germ-line tissue and intestinal cells in addition to pharyngeal expression. Finally, to demonstrate that tropomyosin is essential for development, we inactivated the body wall and pharynx-specific isoforms by RNA-mediated interference. In addition to 50-75 % embryonic lethality in both cases, the worms that survived body wall interference had abnormal body morphology and uncoordinated movements, and those that survived pharynx interference had deformed pharynges and gut regions. These results show the function of tropomyosin in normal muscle filament assembly and embryonic development, and illustrate the different expression patterns characteristic of tropomyosin isoforms in C. elegans.
Assuntos
Processamento Alternativo/genética , Padronização Corporal , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Mucosa Intestinal/metabolismo , Faringe/metabolismo , Tropomiosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Éxons/genética , Expressão Gênica , Regulação da Expressão Gênica , Genes de Helmintos/genética , Genes Reporter/genética , Células Germinativas/metabolismo , Intestinos/citologia , Intestinos/embriologia , Íntrons/genética , Dados de Sequência Molecular , Músculo Liso/metabolismo , Faringe/embriologia , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tropomiosina/análise , Tropomiosina/química , Tropomiosina/genéticaRESUMO
Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles. The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions. Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene. From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin. The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained. The gene is composed of ten short exons encoding a protein of 866 [corrected] amino acid residues. Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods. For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods. However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat. The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods. Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences. Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e. at axial displacements of multiples of 143 A). Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e. at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues. Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments. The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed.
Assuntos
Proteínas de Bactérias/genética , Caenorhabditis/genética , Genes Bacterianos , Tropomiosina/genética , Animais , Bacteriófagos/genética , Sequência de Bases , Caenorhabditis/metabolismo , Clonagem Molecular , Éxons , Immunoblotting , Modelos Biológicos , Dados de Sequência Molecular , MiosinasRESUMO
One of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibatae is the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoichiometric with TF55 (renamed beta). The gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start sites for both alpha and beta were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75 degrees C) to heat shock temperatures (85 to 90 degrees C) indicated that the levels of alpha and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L(-)[35S]methionine at normal and heat shock temperatures indicated significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (> or = 90 degrees C) alpha and beta appeared to be the only two proteins synthesized. The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of alpha and the other of beta. Polyclonal antibodies against alpha and beta from S. shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were aligned with bacterial and eukaryotic chaperonins to generate a phylogenetic tree. The tree reveals the close relationship between the archaeal rosettasomes and the eukaryotic TCP1 protein family and the distant relationship to the bacterial GroEL/HSP60 proteins.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/isolamento & purificação , Sulfolobus/metabolismo , Sequência de Aminoácidos , Animais , Archaea/química , Archaea/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Chaperonina 60/química , Chaperonina 60/genética , Chaperoninas/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Dados de Sequência Molecular , Peso Molecular , Filogenia , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Sulfolobus/genética , Transcrição GênicaRESUMO
The complete tropomyosin gene, designated tmy-1, of Caenorhabditis elegans was recovered by genome walking from a fragment that was obtained by exon-expression cloning using specific cloning using specific anti-tropomyosin antiserum as a probe. The genome structure of the tmy-1 gene has been determined by combining the DNA sequences of cDNA clones with those of the genomic fragments. The single-copy gene spans approximately 13 kb and include 14 exons. Comparison of cDNA and genomic sequences demonstrates that three isoforms are encoded by the gene tmy-1. Homology of the 27 C-terminal amino acid residues to those of Drosophila and vertebrates suggest that these may be the body wall, pharyngeal and non-muscle types. Tissue-specific expression of the tmy-1 gene was determined by microinjection of a promoter/lacZ fusion gene and with immunohistochemistry by using affinity-purified tissue-specific anti-tropomyosins. The 5' end promoter common to CeTMI and CeTMII is expressed in the body wall muscles, vulva, anus muscles and male tail muscles. Control sequences of the 5' end promoter are located 660 to 800 bp upstream of the initial methionine codon. The third isoform, CeTMIII, encoding 256 amino acids residues was expressed in the pharyngeal muscles by the promoter in the third intron. The mRNA of CeTMIII was trans-spliced with SL1 and SL2. These results allow is to solve the question of what is common from this worm to vertebrates, and also what are the cross-species complexities and the tissue-specific differences of tropomyosins. The tmy-1 gene is located on the C. elegans genomic YAC grid near the right end of chromosome I, in the region on the lev-11 gene.
Assuntos
Caenorhabditis elegans/genética , Tropomiosina/química , Tropomiosina/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Caenorhabditis elegans/metabolismo , Mapeamento Cromossômico , Passeio de Cromossomo , Clonagem Molecular , Dosagem de Genes , Expressão Gênica , Genes de Helmintos , Genes Reporter/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Vertebrate reproduction is under the neuroendocrine control of the hypothalamic decapeptide GnRH which synchronizes various reproductive events and influences other reproduction related aspects like spawning behavior and pheromonal action in fish. Multiple forms of GnRH peptides have been reported across diverse vertebrate and invertebrate classes. Here we report the partial seabream GnRH (sbGnRH) cDNA sequence cloned from the brain of Channa striatus (snake head murrel) a fresh water perciform with immense economic and medicinal value across Asiatic countries. sbGnRH mRNA was found in brain, gill and ovary of mature murrel with possible implications to the effect of GnRH on pheromonal phenomena and on reinitiation of oocyte meiosis. In keeping with the earlier reported role of GnRH in initiation of oocyte meiosis we here present evidence from RT-PCR, ICC demonstrating an increase in the level of sbGnRH mRNA in ovary from pre-vitellogenic to post-vitellogenic follicles.