Resumo Este estudo teve como objetivo analisar os níveis de FGF-2 no fluido crevicular peri-implantar durante a terapia de suporte da mucosite. Vinte e seis participantes com prótese protocolo Branemark foram divididos em dois grupos: o grupo controle, caracterizado por saúde peri-implanter, e o grupo mucosite, apresentando diagnóstico de mucosite peri-implantar. Todos os participantes foram submetidos a exame clínico, análise radiográfica, retirada da prótese e terapia não invasiva peri-implantar (debridamento mecânico associado à clorexidina 0,12%) durante um período de 36 dias, dividido em três intervalos. Amostras de fluido crevicular peri-implantar foram coletadas em cada intervalo para análise dos níveis de FGF-2, por ensaio imunoenzimático. Os grupos controle e mucosite não apresentaram diferença nos parâmetros clínicos, exceto para mucosa queratinizada. Ao longo dos intervalos de tratamento, os participantes foram diagnosticados em diferentes grupos, indicando se a terapia não invasiva era ou não capaz de tratar a mucosite peri-implantar. Houve diferença significativa dos níveis de FGF-2 entre os grupos, sendo os níveis de FGF-2 maiores no grupo controle (p = 0.01). Após a terapia de suporte, o grupo com mucosite apresentou níveis de FGF-2 significativamente aumentados (p <0.01) em comparação aos níveis iniciais. Após 36 dias de terapia de suporte, houve redução da mucosite peri-implantar de 70% para 23%. Os resultados clínicos e laboratoriais mostraram uma correlação clara, uma vez que os níveis de FGF-2 aumentaram após 36 dias. O protocolo de terapia foi eficaz e promoveu uma reação regenerativa. O FGF-2 pode ser considerado um alvo futuro para o tratamento da mucosite peri-implantar.

Abstract This study aimed to analyze Fibroblast Growth Factor-2 (FGF-2) levels in the peri-implant crevicular fluid throughout supportive mucositis therapy. Twenty-six participants with Branemark protocol prosthesis were divided into two groups: the control group, characterized by healthy peri-implants, and the mucositis group, presenting a diagnosis of peri-implant mucositis. All participants underwent clinical examination, radiographic analysis, prosthesis removal, and non-invasive peri-implant therapy (mechanical debridement associated with chlorhexidine 0.12%) during a period of 36 days divided into three intervals. Peri-implant crevicular fluid samples were collected at each interval in order to analyze FGF-2 levels by immuno-enzymatic assay. The control and mucositis groups showed difference in keratinized mucosa. The smaller the range of keratinized mucosa the higher susceptibility of peri-implant mucositis. Throughout the treatment intervals, participants were diagnosed in different groups indicating whether or not the non-invasive therapy was able to treat peri-implant mucositis. There was a significant difference of FGF-2 levels between groups, with the higher FGF-2 levels in the control group (p=0.01). After supportive therapy, the mucositis group showed significantly increased FGF-2 levels (p<0.01) compared to initial levels. After 36 days of supportive therapy, there was a reduction of peri-implant mucositis from 70% to 23%. Clinical and laboratory outcomes showed a clear correlation since FGF-2 levels increased after 36 days. It was concluded that the therapy protocol was effective and promoted a regenerative reaction and FGF-2 can be considered a future target for peri-implant mucositis understanding.

Abstract This study determined the effect of thiourethane-functionalized fillers (TU) on the antimicrobial properties, cytotoxicity, degree of conversion (DC), water sorption (Wsp) and solubility (Wsl) of experimental composites. TU-modified fillers were added at different ratios in experimental composites: 0 (Control-TU0), 25% (TU25), 50% (TU50), 75% (TU75) and 100wt% (TU100). The antimicrobial properties were detected through the exhaustion test and counting of Streptococus mutans colonies for biofilm formation. Cytotoxicity to human gingival fibroblasts was evaluated in three different parameters: XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide), NRU (Neutral Red Uptake assay) and CVDE (Crystal Violet Dye Exclusion test)) at the same cells. ELISA was used to measure the IL-6 and b-FGF biomarkers. DC was determined by Fourier-transformed infrared spectroscopy, while Wsp and Wsl by mass variations. Inhibitory capacity of biofilm formation was not observed for any material. All groups presented at least 70% of cell survival within the observed periods (24h and 7 days). Positive control (toxic) had high IL-6 values and low b-FGF values. No significant variations in DC, Wsp, and Wsl were observed among the experimental groups. The use of thiourethane did not present antimicrobial and cytotoxic activity and the tested materials presented equivalent properties to those conventionally used in dentistry.

Abstract Access to genetic resources (GR) and/or traditional knowledge associated with genetic resources (ATK) has been regulated in Brazil since 2001. The law 13,123 / 2015 determined a significant change in the theme, mainly on the rules of distribution of benefits obtained for conservation and sustainable use of biodiversity, the access to technology and technology transfer, the exploitation of products or reproductive material from the GR or ATK and consignment to the outside of part or all the living or dead organism shipped for GR. The implementation of international treaties on GR and ATK for research, biotechnological development and bioprospecting have been causing difficulties for Brazilian researchers, mainly due to the lack of information and dissemination available for compliance with the legislation. In this work, the members of the Committee for Access to Genetic Resources and Associated Traditional Knowledge of the Federal Fluminense University (UFFGEN) - Brazil, and collaborators performed a critical reflection on the new law, helping Brazilian researchers with information necessary to understand the changes made by the new legislation, especially in the field of Biotechnology associated with Brazilian Biodiversity.

Resumo Este estudo teve como objetivo comparar a citotoxicidade dos materiais restauradores indiretos Vita AC12, Lava Ultimate, Vita Enamic e InSync. Extratos de cada material foram preparados por incubação por 1, 7 e 40 dias, com lavagem diária. Fibroblastos gengivais humanos foram expostos aos extratos e a viabilidade celular foi medida por avaliação sequencial da atividade mitocondrial (XTT), integridade da membrana (NRU) e densidade celular (CVDE). Extratos de esferas de poliestireno e fragmentos de látex foram utilizados como controles negativos e positivos, respectivamente. As diferenças entre os grupos e os tempos experimentais foram avaliadas por análise de variância. Na extração de 24 h, observaram-se diferenças significativas entre o controle e Vita AC-12 e InSync no teste do XTT (p<0,05) e entre o controle e os materiais Enamic e Lava Ultimate, no teste CVDE (p<0,05 ). AC12, Lava Ultimate e InSync apresentaram viabilidade celular significativamente menor do que o Enamic e o grupo controle, no ensaio NRU (p<0,05). Os materiais cerâmicos híbridos Vita Enamic e Lava Ultimate apresentaram melhor biocompatibilidade no ponto de tempo de extração de 24 h do que os materiais cerâmicos AC12 e InSync. No entanto, uma simulação de remoção de componentes tóxicos por fluidos biológicos, realizada com o uso de tempos de extração mais prolongados e lavagem diária, levou à ausência de citotoxicidade em todos os materiais restauradores testados. Esses achados podem ser vistos como positivos para a indicação clínica desses materiais restauradores, considerando seu contato com tecidos macios adjacentes por longos períodos de tempo.

Abstract This study aimed to compare the cytotoxicity of the Vita AC12, Lava Ultimate, Vita Enamic and InSync indirect restorative materials. Extracts of each material were prepared by incubation for 1, 7 and 40 days, with daily washing. Human gingival fibroblasts were exposed to the extracts, and cell viability was evaluated by sequential assessment of mitochondrial activity (XTT), membrane integrity (NRU) and cell density (CVDE). Extracts of polystyrene beads and latex fragments were used as negative and positive controls, respectively. Differences between groups and experimental times were evaluated by analysis of variance. At the 24 h extraction, significant differences between the control and both Vita AC-12 and InSync were observed in the XTT assay (p<0.05), and between the control and both Enamic and Lava Ultimate, in the CVDE assay (p<0.05). AC12, Lava Ultimate, and InSync presented significantly lower cell viability than Enamic and the control group, in the NRU assay (p<0.05). The Vita Enamic and Lava Ultimate hybrid ceramic-like materials presented better biocompatibility at the 24 h extraction time point than the AC12 and InSync ceramic materials. However, a simulation of the removal of toxic components by biological fluids, conducted by using longer extraction times and daily washing, led to the absence of cytotoxicity in all the tested restorative materials. These findings can be viewed as positive for the clinical indication of these restorative materials, considering their contact with adjacent soft tissues for extended periods of time.

Resumo Este estudo avaliou comparativamente duas configurações (pó e massa) de um biomaterial composto com base de PLGA (Poli(láctico-co-glicólico)/hidroxiapatita em nanoescala (ReOss®, Intra-Lock International) através da morfologia microscópica, citotoxicidade in vitro, biocompatibilidade e resposta in vivo como substituto ósseo. MEV e EDS caracterizaram os biomateriais antes/após o enxerto. A citocompatibilidade foi avaliada em pré-osteoblastos murinos. A osteocondutividade e a biocompatibilidade foram avaliadas em coelhos Branco da Nova Zelândia. Ambas as configurações foram implantadas na calvária de dezoito animais em defeitos não-críticos, com coágulo sanguíneo como grupo controle. Após 30, 60 e 90 dias, os animais foram eutanasiados e os fragmentos contendo os biomateriais e controles coletados. Blocos ósseos foram embebidos em parafina (n=15) destinados às análises histológica e histomorfométrica, e em resina (n=3) destinadas à MEV e EDS. Antes da implantação, a configuração massa mostrou ambas fases morfológicas porosa e fibrosa. O pó revelou partículas porosas com granulometria variável. EDS mostrou cálcio, carbono e oxigênio na configuração massa, enquanto o pó mostrou também fósforo. Após a implantação a EDS revelou cálcio, carbono e oxigênio em ambas configurações. Os materiais foram considerados citotóxicos pelo teste XTT. A análise histológica mostrou nova formação óssea e nenhuma reação inflamatória nos sítios de implante. Entretanto, a análise histomorfométrica indicou que a quantidade de osso neoformado não foi estatisticamente diferente entre os grupos experimentais. Embora ambos os materiais tenham apresentado citotoxicidade in vitro, foram biocompatíveis e osteocondutores. A configuração do ReOss® afetou as características morfológicas e a citocompatibilidade in vitro, porém não impactou a resposta biológica in vivo, como medido pelo presente modelo.

Article This study evaluated comparatively two configurations (powder and putty) of a composite biomaterial based on PLGA (Poly(lactide-co-glycolide)/nanoescale hydroxyapatite (ReOss®, Intra-Lock International) through microscopic morphology, in vitro cytotoxicity, biocompatibility and in vivo response as a bone substitute. SEM and EDS characterized the biomaterials before/after grafting. Cytocompatibility was assessed with murine pre-osteoblasts. Osteoconductivity and biocompatibility were evaluated in White New Zealand rabbits. Both configurations were implanted in the calvaria of eighteen animals in non-critical size defects, with blood clot as the control group. After 30, 60 and 90 days, the animals were euthanized and the fragments containing the biomaterials and controls were harvested. Bone blocks were embedded in paraffin (n=15) aiming at histological and histomorphometric analysis, and in resin (n=3) aiming at SEM and EDS. Before implantation, the putty configuration showed both a porous and a fibrous morphological phase. Powder revealed porous particles with variable granulometry. EDS showed calcium, carbon, and oxygen in putty configuration, while powder also showed phosphorus. After implantation EDS revealed calcium, carbon, and oxygen in both configurations. The materials were considered cytotoxic by the XTT test. Histological analysis showed new bone formation and no inflammatory reaction at implant sites. However, the histomorphometric analysis indicated that the amount of newly formed bone was not statistically different between experimental groups. Although both materials presented in vitro cytotoxicity, they were biocompatible and osteoconductive. The configuration of ReOss® affected morphological characteristics and the in vitro cytocompatibility but did not impact on the in vivo biological response, as measured by the present model.

Abstract Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. Objective To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). Material and Methods Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. Results While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. Conclusions All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.

Abstract Calcium silicate-based materials have been widely studied due to their resemblance to, and similar applicability of, mineral trioxide aggregate (MTA). Among these, Biodentine™ (BD) was specifically designed as a “dentin replacement” material for applications such as root perforations, apexification, treatment of resorptive lesions, and as a retrograde filling material. The present study aimed to assess the in vitro response of human primary osteoblasts to BD using MTA AngelusTM as a reference material, by simultaneously analyzing three different cell viability parameters, namely mitochondrial activity, membrane integrity, and cell density. BD and MTA extracts were prepared by incubation on culture media for 24 h or 42 days after mixing. Primary human osteoblasts were exposed to extracts for 24 h, at 37oC with 5% CO2, and cell viability was evaluated by the XTT, NRU, and CVDE assays. Both materials induced cell viability levels higher than 70% when extracted for 24 h. However, when cells were exposed to extracts with increased conditioning times, MTA presented significant cytotoxic effects (p < 0.05) in comparison to the control and MTA at 24 h. After 42 days, the XTT assay identified a significant reduction in cell viability by BD when compared to the control (p<0.05), despite the fact that levels above the 70% viability cutoff were attained for biocompatible materials. It can be concluded that BD is cytocompatible with human primary osteoblasts, indicating its adequacy in direct contact with bone tissues.

Abstract Dental caries is an oral pathology associated with both lifestyle and genetic factors. The caries process can be influenced by salivary composition, which includes ions and proteins. Studies have described associations between salivary protein polymorphisms and dental caries experience, while others have shown no association with salivary proteins genetic variability. The aim of this study is to assess the influence of salivary protein polymorphisms on the risk of dental caries by means of a systematic review of the current literature. An electronic search was performed in PubMed, Scopus, and Virtual Health Library. The following search terms were used: “dental caries susceptibility,” “dental caries,” “polymorphism, genetics,” “saliva,” “proteins,” and “peptides.” Related MeSH headings and free terms were included. The inclusion criteria comprised clinical investigations of subjects with and without caries. After application of these eligibility criteria, the selected articles were qualified by assessing their methodological quality. Initially, 338 articles were identified from the electronic databases after exclusion of duplicates. Exclusion criteria eliminated 322 articles, and 16 remained for evaluation. Eleven articles found a consistent association between salivary protein polymorphisms and risk of dental caries, for proteins related to antimicrobial activity (beta defensin 1 and lysozyme-like protein), pH control (carbonic anhydrase VI), and bacterial colonization/adhesion (lactotransferrin, mucin, and proline-rich protein Db). This systematic review demonstrated an association between genetic polymorphisms and risk of dental caries for most of the salivary proteins.

Resumo Resinas bisacrílicas são usadas em restaurações dentárias provisórias e têm mostrado vantagens em relação a outros materiais. O objetivo deste trabalho foi avaliar a citotoxicidade in vitro de duas resinas compostas bisacrílicas (Protemp 4 e Luxatemp Star), obtidas após 1, 7 e 40 dias da mistura com os componentes da resina, usando um ensaio padronizado empregando células primárias humanas fortemente relacionadas aos tecidos orais. Culturas de células de fibroblastos gengivais humanos foram expostas por 24 h aos extratos das resinas bisacrílicas, esferas de poliestireno (controle negativo) e látex (controle positivo), obtidos após diferentes períodos de incubação, a 37 °C e com 5% CO2. A viabilidade celular foi avaliada usando procedimentos multiparamétricos que envolvem a avaliação sequencial (usando as mesmas células) da atividade mitocondrial (ensaio XTT), a integridade de membrana (teste do vermelho neutro) e a densidade celular total (teste de exclusão do corante cristal violeta). As células expostas aos extratos mostraram viabilidade celular acima de 75% depois de 24 h. Mesmo quando as células foram expostas aos extratos com aumento do tempo de condicionamento, as resinas bisacrílicas não apresentaram efeitos citotóxicos significativos (p<0,05), comparadas ao grupo controle ou em relação às primeiras 24 h de contato com os produtos. Não houve diferença entre os resultados obtidos para as resinas bisacrílicas avaliadas entre as 24 h, 7 e 40 dias depois da mistura. Concluímos que as resinas bisacrílicas Protemp 4 e Luxatemp Star foram citocompatíveis com os fibroblastos gengivais humanos, sugerindo que ambos materiais são adequados para uso em contato com tecidos humanos.

Abstract Bis-acryl resins are used for temporary dental restorations and have shown advantages over other materials. The aim of this work was to evaluate the in vitro cytotoxicity of two bis-acryl composite resins (Protemp 4 and Luxatemp Star), obtained at 1, 7 and 40 days after mixing the resin components, using a standardized assay employing human primary cells closely related to oral tissues. Human gingival fibroblast cell cultures were exposed for 24 h to either bis-acryl composite resins, polystyrene beads (negative control) and latex (positive control) extracts obtained after incubation by the different periods, at 37 °C under 5% CO2. Cell viability was evaluated using a multiparametric procedure involving sequential assessment (using the same cells) of mitochondrial activity (XTT assay), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). The cells exposed to the resin extracts showed cell viability indexes exceeding 75% after 24 h. Even when cells were exposed to extracts prepared with longer conditioning times, the bis-acryl composite resins showed no significant cytotoxic effects (p>0.05), compared to the control group or in relation to the first 24 h of contact with the products. There were no differences among the results obtained for the bis-acryl composite resins evaluated 24 h, 7 days and 40 days after mixing. It may be concluded that the bis-acryl resins Protemp 4 and Luxatemp Star were cytocompatible with human gingival fibroblasts, suggesting that both materials are suitable for use in contact with human tissues.

ABSTRACT Objective The aim of this study was to investigate the in vitro and in vivo biological responses to nanostructured carbonated hydroxyapatite/calcium alginate (CHA) microspheres used for alveolar bone repair, compared to sintered hydroxyapatite (HA). Material and Methods The maxillary central incisors of 45 Wistar rats were extracted, and the dental sockets were filled with HA, CHA, and blood clot (control group) (n=5/period/group). After 7, 21 and 42 days, the samples of bone with the biomaterials were obtained for histological and histomorphometric analysis, and the plasma levels of RANKL and OPG were determined via immunoassay. Statistical analysis was performed by Two-Way ANOVA with post-hoc Tukey test at 95% level of significance. Results The CHA and HA microspheres were cytocompatible with both human and murine cells on an in vitro assay. Histological analysis showed the time-dependent increase of newly formed bone in control group characterized by an intense osteoblast activity. In HA and CHA groups, the presence of a slight granulation reaction around the spheres was observed after seven days, which was reduced by the 42nd day. A considerable amount of newly formed bone was observed surrounding the CHA spheres and the biomaterials particles at 42-day time point compared with HA. Histomorphometric analysis showed a significant increase of newly formed bone in CHA group compared with HA after 21 and 42 days from surgery, moreover, CHA showed almost 2-fold greater biosorption than HA at 42 days (two-way ANOVA, p<0.05) indicating greater biosorption. An increase in the RANKL/OPG ratio was observed in the CHA group on the 7th day. Conclusion CHA spheres were osteoconductive and presented earlier biosorption, inducing early increases in the levels of proteins involved in resorption.

Objective The aim of this study was to produce dense granules of tricalcium phosphate (β-TCP) and magnesium (Mg) substituted β-TCP, also known as β-TCMP (Mg/Ca=0.15 mol), in order to evaluate the impact of Mg incorporation on the physicochemical parameters and in vitro biocompatibility of this novel material. Material and Methods The materials were characterized using X-ray diffraction (XRD), infrared spectroscopy (FTIR), electron microscopy and inductively coupled plasma (ICP). Biocompatibility was assayed according to ISO 10993-12:2007 and 7405:2008, by two different tests of cell survival and integrity (XTT and CVDE). Results The XRD profile presented the main peaks of β-TCP (JCPDS 090169) and β-TCMP (JCPDS 130404). The characteristic absorption bands of TCP were also identified by FTIR. The ICP results of β-TCMP granules extract showed a precipitation of calcium and release of Mg into the culture medium. Regarding the cytotoxicity assays, β-TCMP dense granules did not significantly affect the mitochondrial activity and relative cell density in relation to β-TCP dense granules, despite the release of Mg from granules into the cell culture medium. Conclusion β-TCMP granules were successfully produced and were able to release Mg into media without cytotoxicity, indicating the suitability of this promising material for further biological studies on its adequacy for bone therapy.

The aim of this work was to evaluate the effects of different times of extraction on the cytotoxicity of six representatives of different root canal sealer groups-Real Seal SE, AH Plus, GuttaFlow, Sealapex, Roth 801, and ThermaSeal Plus-with human gingival fibroblasts. The materials were prepared according to manufacturers' specifications, and were incubated in culture medium (DMEM) at 37ºC for 1, 7, 14, 21, and 28 days, with daily washing, to simulate periodontal ligament clearance. Human fibroblasts were exposed to the final extracts at 24 hours, and cell viability was determined by MTT assay, with exposure to unconditioned DMEM as a negative control. Statistical analysis comparing cytotoxicities at each exposure time was performed by ANOVA with Scheffé adjustment for multiple comparisons at a 95% confidence level. Results indicated that GuttaFlow was significantly less cytotoxic than all other sealers (p < 0.05) at 1 day of extraction. After 7 days of extraction, cell viability for GuttaFlow was significantly increased as compared with that of all groups except sealer AH Plus. At day 14, cytotoxicity of Sealapex was significantly higher than that of all other sealers (p < 0.05). At days 21 and 28, there were no significant differences in cytotoxicity among sealer groups. All materials presented some level of cytotoxicity to fibroblasts, while GuttaFlow was the least cytotoxic sealer tested. However, the cytotoxicity of all materials seemed to decrease similarly in a time-dependent manner.

The incorporation of zinc onto biomaterials may improve their biological properties, since it is an essential trace element, involved on important steps of bone remodeling. The aim of this work was to evaluate the in vivo biocompatibility of previously characterized hydroxyapatite granules incorporated with 5% zinc (ZnHA), through a subcutaneous grafting test. Granules produced from powders of ZnHA or stoichiometric HA were subcutaneously grafted onto Balb/c mice, and histomorphometrical analysis was performed at 7, 14, 21, 28 or 60 days after grafting. HA and ZnHA induced a similar foreign body reaction, with formation of granulome and the presence of a fibrous capsule. Granulocytes were virtually absent, and agranulocytes decreased with time. ZnHA treated samples presented faster reduction on multinucleated giant cells and increase on observed angiogenesis, as compared to HA. It could be concluded that ZnHA granules are biocompatible. Further study should better address its potential use for bone therapy.

A atividade da fosfofrutocinase (PFK) de Rodnius prolixus, um inseto hematófago, o qual vôa somente pequenas distâncias, foi medida e comparada em dois tecidos metabolicamente ativos - músculo de asa e corpo gorduroso. A atividade desta importante enzima glicolítica regulatória foi muito mais pronunciada em músculo de asa (15,1 ±1,4 U/mg) do que em extrato de corpo gorduroso (3,6 ±0,4 U/mg) embora este último tenha apresentado níveis mais altos da enzima por quantidade de proteína, como medido por western-blotting. Extratos de músculo foram mais responsivos do que corpo gorduroso para ATP e frutose-6-fosfato, ambos substratos da PFK. A regulação alostérica exercida por diferentes efetores tais como ADP, AMP, frutose-2,6-bisfosfato apresentou um padrão singular para cada tecido. O pH ótimo (8,0-8,5) e a sensibilidade a variações de pH, foram muito similares e o citrato foi incapaz de inibir a atividade da PFK em ambos os extratos. Nossos resultados sugerem a existência de uma atividade particular da PFK para cada tecido com padrões regulatórios que são consistentes com suas funções fisiológicas.

6-phosphofructo-1-kinase (phosphofructokinase; PFK) activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues - flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 ± 1.4 U/mg) than in fat body extracts (3.6±0.4 U/mg), although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5) and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.

Neste trabalho, nós relatamos evidencias de que a associação entre fosfofrutocinase e F-actina pode ser afetada por estímulos pela insulina em homogeneizados de músculo esquelético de coelho e que esta associação pode ser um mecanismo de regulação da fosfofrutocinase. Através de tecnicas de co-sedimentação, nós observamos que em tecidos estimulados pela insulina, aproximadamente 70% da atividade fosfofrutocinásica esta co-localizada a fração enriquecida com actina, contra 28% no controle. Este fenômeno é acompanhado por um aumento de 100% na atividade específica da fosfofrutocinase em homogeneizados estimulados. F-actina purificada causou um aumento de 230% na atividade da fosfofrutocinase e alterou seus parâmetros cinéticos. A presença de F-actina aumentou a afinidade da fosfofrutocinase pela frutose 6-fosfato, sem alterar, no entanto, a velocidade máxima (Vmax). Nós propomos que a modulação da distribuição celular da fosfofrutocinase pode ser um dos mecanismos de controle do fluxo glicolítico em músculos de mamíferos pela insulina.

In this work, we report evidences that the association of phosphofructokinase and F-actin can be affected by insulin stimulation in rabbit skeletal muscle homogenates and that this association can be a mechanism of phos-phofructokinase regulation. Through co-sedimentation techniques, we observed that on insulin-stimulated tissues, approximately 70% of phosphofructokinase activity is co-located in an actin-enriched fraction, against 28% in control. This phenomenon is accompanied by a 100% increase in specific phosphofructokinase activity in stimulated homogenates. Purified F-actin causes an increase of 230% in phosphofructokinase activity and alters its kinetic parameters. The presence of F-actin increases the affinity of phosphofructokinase for fructose 6-phosphate nevertheless, with no changes in maximum velocity (Vmax). Here we propose that the modulation of cellular distribution of phosphofructokinase may be one of the mechanisms of control of glycolytic flux in mammalian muscle by insulin.