RESUMO
Obesity is a metabolic disease characterized by low-level chronic inflammation. Obese individuals are susceptible to infection by viruses, and vaccination against these pathogens is less effective than in nonobese individuals. Here, we sought to explore the immunological environment in a mouse model of obesity induced by a high-fat diet (HFD). HFD treatment increased the body weight and epididymal fat mass. The proportion of activated B cells, T cells, and macrophages was similar between mice in the HFD group and the regular-fat diet (RFD) group. The Th1 cell subpopulation in the HFD group was increased, whereas the proportion of Treg cells was reduced compared with the RFD group. Moreover, T-cell proliferation and cytokine production did not differ between the groups when cells were stimulated with anti-CD3 and anti-CD28 antibodies in vitro. In macrophages, phagocytic activity was higher in mice fed an HFD than in those fed an RFD, but expression levels of CD86 and MHC class II antigens were similar. When macrophages were cultured in vitro, the proportion of CD86-expressing macrophages was lower in those isolated from mice in the HFD group than in those isolated from the RFD group. Furthermore, lipopolysaccharide-induced interleukin 6 (IL-6) and tumor necrosis factor alpha secretions were significantly reduced in macrophages isolated from the HFD group. In addition, influenza vaccine-induced antibodies in the HFD group diminished more rapidly than in the RFD group. These results suggest that poor functionality of macrophages during obesity might contribute to a reduction in vaccine efficacy.
Assuntos
Anticorpos Antivirais/sangue , Dieta/efeitos adversos , Gorduras na Dieta/administração & dosagem , Vacinas contra Influenza/imunologia , Macrófagos/fisiologia , Obesidade/imunologia , Animais , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Gorduras na Dieta/efeitos adversos , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologiaRESUMO
In this longitudinal study with Single Comb White Leghorn chickens, we investigated the effects of stress conditions in birds that were subjected to a high stocking density with feed restrictions on the quantity of telomeric DNA, the rate of DNA damage, and the expression levels of heat shock proteins (HSP) and hydroxyl-3-methyl-glutaryl coenzyme A reductase (HMGCR) genes. The telomere length and telomere-shortening rates were analyzed by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes. The DNA damage rate of lymphocytes was quantified by the comet assay. The expression levels of HSP70, HSP90, and HMGCR genes were measured by quantitative real-time PCR in lymphocytes. The telomere-shortening rate of the lymphocytes was significantly higher in the stress group than in the control. The DNA damage also increased in birds raised under stress conditions, as compared with the control group. The stress conditions had a significant effect on the expressions of HMGCR and HSP90α in lymphocytes but had no significance on HSP70 and HSP90ß in blood. We conclude that the telomere length, especially the telomere-shortening rates, the quantification of total DNA damage, and the expression levels of the HMGCR and HSP90α genes can be used as sensitive physiological stress markers in chickens.
Assuntos
Galinhas/fisiologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/metabolismo , Animais , Galinhas/genética , Ensaio Cometa/veterinária , DNA/análise , Dano ao DNA , Privação de Alimentos , Hibridização in Situ Fluorescente/veterinária , Estudos Longitudinais , Linfócitos/metabolismo , Densidade Demográfica , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Telômero/metabolismoRESUMO
This study was conducted to evaluate whether the sex of donor primordial germ cells (PGCs) influences production of chimeric semen from recipient hatchlings produced by interspecies transfer between pheasant (Phasianus colchicus) and chicken (Gallus gallus). Pheasant PGCs were retrieved from 7-d-old embryos and subsequently transferred into circulatory blood of 2.5-d-old (Stage 17) embryos. The sex of embryos was discerned 3 to 6 days after laying, and in preliminary study, overall rate of embryo survival after sexing was 74.6% with male-to-female ratio of 0.49 to 0.51. In Experiment 1, magnetic-activated cell sorting (MACS) using QCR1 antibody was effective for enriching the population of male and female PGCs in gonadal cells (9.2- to 12.5-fold and 10.8- to 19.5-fold increase, respectively). In Experiment 2, an increase in the number of hatchlings producing chimeric semen was detected after the homosexual transfer of male-to-male compared with that after the heterosexual transfer of female-to-male (68% to 88%). Significant increase was found in the frequency of chimeric semen production (0.96 to 1.68 times); production of pheasant progenies by artificial insemination using chimeric semen was also increased in the homosexual transfer (0 to 3 cases). In conclusion, the homosexual PGC transfer of male-to-male yielded better rate of generating pheasant progenies after test cross-reproduction than that of the heterosexual transfer of female-to-male, which could improve the efficiency of interspecies germ cell transfer system.
Assuntos
Quimera/fisiologia , Células Germinativas/transplante , Animais , Animais Geneticamente Modificados , Embrião de Galinha , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Feminino , Galliformes/embriologia , Engenharia Genética , Masculino , Sêmen/citologia , Processos de Determinação Sexual , Fatores Sexuais , Razão de MasculinidadeRESUMO
The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.
Assuntos
Clonagem de Organismos/veterinária , Lobos , Animais , Clonagem de Organismos/métodos , Cães , Extinção Biológica , Feminino , Masculino , Repetições de Microssatélites , Técnicas de Transferência Nuclear , Oócitos , GravidezRESUMO
A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.
Assuntos
Núcleo Celular/genética , Sondas de DNA/metabolismo , Freemartinismo/diagnóstico , Hibridização in Situ Fluorescente/veterinária , Interfase/genética , Linfócitos/citologia , Cromossomo Y/genética , Animais , Bovinos , Feminino , Masculino , Reprodutibilidade dos TestesRESUMO
Several features of the implant surface, such as roughness, topography and composition play a relevant role in implant integration with bone. This study was conducted in order to determine the effects of various thin layer hydroxyapatite (HA) coatings on anodized Ti surfaces on the biological responses of a human osteoblast-like cell line (MG63). MG63 cells were cultured on 100 nm HA (100 nm HA coating on anodized surface), 500-700 nm HA (500-700 nm HA coating on anodized surface), 1 mum HA (1 mum HA coating on anodized surface) and anodize (non-HA coating on anodized surface) Ti. The morphology of these cells was assessed by scanning electron microscopy (SEM). The cDNAs prepared from the total RNAs of the MG63 were hybridized into a human cDNA microarray (1152 elements). The appearances of the surfaces observed by SEM were different on each of the four dental substrate types. MG63 cells cultured on 100 nm HA, 1 mum HA and anodize exhibited cell-matrix interactions. It was 500-700 nm HA surface showing cell-cell interaction. In the expression of genes involved in osseointegration, several genes, including bone morphogenetic protein 2, latent transforming growth factor beta binding protein 1, catenin (cadherin-associated protein), integrin, PDGFRB and GDF-1 growth differentiation factor 1 were up-regulated on the different surfaces. Several genes, including fibroblast growth factor receptor 3, fibroblast growth factor 12 and CD4 were down-regulated on the different surfaces. The attachment and expression of key osteogenic regulatory genes were enhanced by the surface morphology of the dental materials used.
Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Implantes Dentários , Durapatita/farmacologia , Osteoblastos/efeitos dos fármacos , Titânio/química , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Teste de Materiais/métodos , Microscopia Eletrônica de Varredura , Osseointegração/genética , Osteoblastos/ultraestrutura , Propriedades de SuperfícieRESUMO
During the process of bone formation, titanium (Ti) surface is an important factor in the modulation of osteoblastic function. This study was conducted in order to determine the effects of different Ti surfaces on the biological responses of a human osteoblast-like cell line (MG63). MG63 cells were cultured on smooth (S), sandblasted large-grit and acid etching (SLA), hydroxyapatite (HA), hydroxyfluoride (HF), titanium nitrate (TIN), and diamond-like carbon (DLC) Ti. The morphology of these cells were assessed by SEM. The cDNAs prepared from the total RNAs of the MG63 were hybridized into a human cDNA microarray (1152 elements). The appearances of the surfaces observed by SEM were different on each of the six dental substrate types. The SLA and HA surfaces were determined to be rougher than the others. MG63 cells cultured on SLA and HA exhibited cell-matrix interactions. In the expression of genes involved in osseointegration, several genes, including bone morphogenetic protein, cadherin, integrin, and insulin-like growth factors, were upregulated on the different surfaces. Several genes, including fibroblast growth factor receptor 4, Bcl 2-related protein, and collagen, were downregulated on the different surfaces. The attachment and expression of key osteogenic regulatory genes were enhanced by the surface roughness of the dental materials used.
Assuntos
Implantes Dentários , Osteoblastos/fisiologia , Titânio , Apoptose/genética , Adesão Celular/genética , Comunicação Celular/genética , Proliferação de Células , Materiais Revestidos Biocompatíveis , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Teste de Materiais/métodos , Microscopia Eletrônica de Varredura , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Osseointegração/genética , Osteoblastos/ultraestrutura , Propriedades de SuperfícieRESUMO
Molecular genetic techniques were used to determine if mutations in the genes encoding anti-Müllerian hormone (AMH) (also known as Müllerian inhibiting substance (MIS)) and its receptor (AMHR) are commonly present in patients with congenital absence of the uterus and vagina (CAUV). Twenty-two CAUV patients and 96 control subjects from diverse ethnic groups were studied after obtaining informed consent. Genomic DNA samples prepared from leukocytes were digested separately with several different restriction enzymes, and the resultant fragments were analyzed for restriction fragment melting polymorphisms (RFMPs) by denaturing gradient gel electrophoresis (DGGE). Electrophoretic mobility of DNA fragments which were 200-700 base pairs in length was compared using polyacrylamide gels that included linear gradients of denaturing solvents designed to separate DNA fragments according to sequence-dependent variation in thermal stability. Two RFMPs were found in the AMH gene in both patients and normal control subjects. One RFMP in the AMHR gene was present at low frequencies in both patients and normal control subjects. No RFMPs specific to CAUV patients were found in either gene. Because no mutations or rare DNA sequence polymorphisms were detected in the AMH and the AMHR genes in this group of CAUV patients, it is unlikely that either gene commonly has an etiologic role in CAUV.
Assuntos
Glicoproteínas , Inibidores do Crescimento/genética , Receptores de Peptídeos/genética , Hormônios Testiculares/genética , Anormalidades Urogenitais/genética , Útero/anormalidades , Vagina/anormalidades , Hormônio Antimülleriano , DNA/análise , Feminino , Humanos , Ductos Paramesonéfricos/crescimento & desenvolvimento , Mutação , Polimorfismo de Fragmento de Restrição , Receptores de Fatores de Crescimento Transformadores betaRESUMO
Deoxycytidylate hydroxymethylase from bacteriophage T4 is a homodimeric enzyme in which each polypeptide chain consists of 246 amino-acid residues. It has been crystallized in the presence of its substrate, deoxycytidine monophosphate, at room temperature using sodium citrate as precipitant. The crystals are monoclinic, belonging to space group C2, with unit-cell parameters a = 174.22, b = 53.12, c = 75.17 A, beta = 115.29 degrees. The asymmetric unit contains one homodimer, with a corresponding Vm of 2.65 A3 Da-1 and solvent content of 54%. Native diffraction data to 1.6 A resolution have been collected from two crystals using synchrotron radiation.
Assuntos
Bacteriófago T4/enzimologia , Hidroximetil e Formil Transferases/química , Catálise , Cristalização , Cristalografia por Raios X , Hidroximetil e Formil Transferases/isolamento & purificação , Hidroximetil e Formil Transferases/metabolismo , Luz , Peso Molecular , Espalhamento de Radiação , Especificidade por SubstratoRESUMO
Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built.
Assuntos
Bacteriófago T4/enzimologia , Hidroximetil e Formil Transferases/química , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Desoxicitidina Monofosfato/química , Dimerização , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Timidilato Sintase/química , Proteínas Virais/químicaRESUMO
OBJECTIVE: To compare the efficacy of two clinically accepted methods of progesterone supplementation during IVF. DESIGN: Prospective randomized trial. SETTING: A university-based IVF program. PATIENT(S): Three hundred fourteen stimulated IVF cycles between January 1993 and October 1994. INTERVENTION(S): Patients were assigned to one of two luteal phase progesterone regimens by a random permuted block design. In protocol A, 12.5 mg of IM progesterone was given 12 hours before oocyte retrieval; in protocol B, 25 mg of IM progesterone was given on the day of oocyte retrieval. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Patient demographic characteristics, including age, diagnosis, number of oocytes retrieved and fertilized, and number of embryos transferred, were not different between the two groups. There was no difference in the rate of cycle cancellation between the groups. One hundred forty ETs were performed in patients assigned to protocol A and 142 in patients assigned to protocol B. The clinical pregnancy rate in group A was 12.9% compared with 24.6% in group B. CONCLUSION(S): The administration of progesterone before oocyte retrieval is associated with a lower pregnancy rate than the administration of progesterone after oocyte retrieval.
Assuntos
Implantação do Embrião/efeitos dos fármacos , Doação de Oócitos , Progesterona/efeitos adversos , Adulto , Gonadotropina Coriônica/farmacologia , Método Duplo-Cego , Transferência Embrionária , Feminino , Humanos , Ovulação/efeitos dos fármacos , Ovulação/fisiologia , Gravidez , Estudos Prospectivos , Resultado do TratamentoRESUMO
OBJECTIVE: To determine if GnRH receptor mutations occur in patients with idiopathic hypogonadotropic hypogonadism. DESIGN: Patients and controls were studied by molecular genetic analysis. SETTING: A tertiary medical center setting. PATIENT(S): Twenty-four patients with idiopathic hypogonadotropic hypogonadism and 20 controls. INTERVENTION(S): Deoxyribonucleic acid from all individuals was analyzed by Southern blot analysis and denaturing gradient gel electrophoresis. Genomic DNA was digested with restriction enzymes, and Southern blots and denaturing gradient gel blots were constructed. Blots were hybridized with the GnRH receptor complementary DNA probe. The DNA sequencing was performed on samples from two representative patients. MAIN OUTCOME MEASURE(S): Gonadotropin-releasing hormone receptor gene structure was ascertained by comparing fragments from autoradiographs in patients and controls. Individual nucleotides were ascertained from DNA sequencing gels. RESULT(S): No GnRH receptor gene deletions or polymorphisms were identified by Southern blot analysis. New restriction-fragment melting polymorphisms using the enzymes DpnII, RsaI, and HaeIII were identified by denaturing gradient gel blots in patients and controls. CONCLUSION(S): Gonadotropin-releasing hormone receptor gene deletions or rearrangements were not observed in our idiopathic hypogonadotropic hypogonadism patients. Denaturing gradient gel electrophoresis failed to identify single-base differences unique to patients with idiopathic hypogonadotropic hypogonadism, dramatically reducing the likelihood that point mutations of the GnRH receptor gene are present in idiopathic hypogonadotropic hypogonadism.
Assuntos
Gonadotropinas Hipofisárias/sangue , Hipogonadismo/genética , Mutação Puntual , Receptores LHRH/genética , Adolescente , Adulto , Autorradiografia , Southern Blotting , Estudos de Casos e Controles , Primers do DNA , DNA Complementar , Eletroforese em Gel de Ágar , Feminino , Humanos , Hipogonadismo/sangueRESUMO
Fifty breasts with nonpalpable ductal carcinoma in situ (DCIS) were examined for the presence of microinvasion, multicentricity, and number of involved ducts to see if the biopsy specimen could have predicted the findings in the remainder of the breast. When DCIS was an incidental finding, fewer ducts were involved and no evidence of either microinvasion or multicentricity was found. Solid and cribriform DCIS were rarely multicentric or microinvasive; micropapillary DCIS was often multicentric, rarely microinvasive; comedocarcinoma was more likely to be both microinvasive and multicentric. Ductal carcinoma in situ as an incidental finding may be treated by excision alone; papillary and micropapillary DCIS are best treated by therapy aimed at the entire breast, although axillary dissection may not be required. Therapy for comedocarcinomas should include the entire breast and the axillary nodes.