RESUMO
The growth of cyanobacteria can vary considerably depending on the ambient temperature. Since the optimal growth temperature for Arthrospira platensis (strain SAG21.99) is not yet known, this was investigated in the present study. The study revealed that a process temperature of 30 °C seems to be optimal for the Arthrospira strain SAG21.99 cultivation in terms of a maximum biomass productivity. This was also true for the phycocyanin content which was at 30 °C significantly higher than at 20 or 40 °C.
Assuntos
Spirulina , TemperaturaRESUMO
Arthrospira platensis (AP) and some of its derived products have well-established biological activities as antioxidants or as agents to reduce cardiovascular disease risk factors. Furthermore, AP products have gained increasing importance as potential anti-cancer agents. However, the ingredients of the available products vary greatly with the origin, the type of production and processing, which could have significant consequences for their biological effects. Therefore, the composition and biological influence of five distinct AP powders, which were acquired commercially or produced at a public biotechnology institute, were investigated in regard to their endothelialization capacity using a cell impedance- (CI) based measurement method. The study revealed that the AP composition and especially the influence on HUVEC proliferation differed significantly between the five AP powders up to 109%.Thus, it could be shown that the method used allows the reliable detection of quantitative differences in biological effects of different AP preparations.
Assuntos
Spirulina , Antioxidantes , Células Endoteliais , PósRESUMO
Within the last years a comprehensive number of scientific studies demonstrated beneficial effect of Arthropira platensis (AP) as dietary supplement due to a high content of proteins, minerals and vitamins. Positive effects like promoting the immune system, reducing inflammation and an anti-oxidant capacity are reported. In this study, the effect of an aqueous AP extract on primary human venous endothelial cells (HUVEC) was investigated. In addition, the effect of AP on HUVEC treated with a bacterial toxin (lipopolysaccharide, LPA), inducing an activation of HUVEC and cellular detachment, was analyzed. Depending on the concentration of AP extract a significantly accelerated formation of an endothelial cell monolayer was observed. Furthermore, the detachment of HUVEC after LPA addition was dramatically reduced by AP. In conclusion, the data are promising and indicatory for an application of Arthrospira platensis in the clinical field.
Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Ficocianina/farmacologia , Probióticos/farmacologia , Spirulina/química , Endotélio Vascular/patologia , Humanos , Técnicas In Vitro , Estudos ProspectivosRESUMO
BACKGROUND: In the early phase of the COVID-19 pandemic Germany missed to set up efficient containment measures. Consequently, the number of cases increased exponentially until a lockdown was implemented to suppress the spread of SARS-CoV-2. Fortunately, Germany has a high capability for coronavirus lab testing and more than 30,000 ICU beds. These capabilities and the lockdown turned out to be an advantage to combat the pandemic and to prevent a health-system overload. AIM: The aim was to predict the plateau day of SARS-CoV-2 infections or deaths. RESULTS: The effect on the viral spread of the German measures taken and the impact on the peak of new infection cases is shown. By normalizing daily case numbers, the plateau day of the current outbreak in Germany could be calculated to be reached at April 12, 2020 (day 103 of 2020). CONCLUSION: Normalized case number curves are helpful to predict the time point at which no further new infections will occur if the epidemic situation remains stable. Upon reaching the plateau day during a lockdown phase, a residual time-period of about 2-3 weeks can be utilized to prepare a safe unlocking period. As can be learned from Asian countries such as South Korea and Taiwan there must be strict rules to keep the risk of infection low. Those include social distancing, face mask wearing in combination with digital contact tracing and serosurveillance studies. Following those rules, a safe dance around the infection curve allows to keep the population at a reduced infection rate.
Assuntos
Controle de Doenças Transmissíveis/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/terapia , Pneumonia Viral/epidemiologia , Pneumonia Viral/terapia , Betacoronavirus , COVID-19 , China/epidemiologia , Infecções por Coronavirus/prevenção & controle , Surtos de Doenças , Alemanha/epidemiologia , Humanos , Infectologia/tendências , Unidades de Terapia Intensiva , Modelos Lineares , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Quarentena , SARS-CoV-2 , Organização Mundial da SaúdeRESUMO
Some months ago, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan, China, and spread rapidly around the world. Some states, such as the Netherlands, Germany, Great Britain, Sweden and the USA initially focused on keeping the restrictions for economy and society as low as possible. The responsible authorities were of the opinion - and still are e.g. in Sweden - that it is sufficient enough to protect particularly vulnerable persons such as the elderly or people with pre-existing conditions. The idea behind this is that as soon as 60 to 70 percent of the population is infected with a pathogen, a so-called "herd immunity" has developed. However, the increasing numbers of deaths and modelling studies showed the expected overload of the hospitals. Therefore, most countries decided for a temporary lockdown with the exception of Sweden.Based on the number of the total population, three times more people died from COVID-19 in Sweden (2679 deaths per 10 million inhabitants) compared to Germany (6848 deaths per 80 million inhabitants). The comparison Sweden versus Taiwan is even worse because 1072 times more people died in Sweden based on the number of the population (6 deaths per 24 million inhabitants).In the face of the lack of an antiviral treatment and the lack of a protective vaccine one must state Taiwan has made the best out of the pandemic situation whereas Sweden failed completely.
Assuntos
Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , COVID-19 , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Feminino , Humanos , Imunidade Coletiva , Masculino , Pandemias/prevenção & controle , Pneumonia Viral/patologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , SARS-CoV-2RESUMO
There is growing evidence that COVID-19 not only affects the lungs but beyond that the endothelial system. Recent studies showed that this can lead to microcirculatory impairments and in consequence to functional disorders of all inner organs. The combination of endothelial dysfunction with a generalized inflammatory state and complement elements may together contribute to the overall pro-coagulative state described in COVID-19 patients leading to venular as well as to arteriolar occlusions.
Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/patologia , Endotélio Vascular/virologia , Pneumonia Viral/patologia , COVID-19 , Infecções por Coronavirus/virologia , Endotélio Vascular/patologia , Humanos , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2RESUMO
Endothelialization of cardiovascular implants is regarded as a promising strategy for long-term compatibility. While umbilical vein endothelial cells are typically applied in research, human arterial endothelial cells (HAEC) from elderly donors would be the obvious source for autologous cellularization strategies.In our approach, HAEC from 16 donors of varying age (16-63 years) were divided into two groups (<30 years and >30 years) and analyzed regarding morphology, viability, proliferation, function and senescence status.No age-related differences were found regarding morphology, viability, density, prostacyclin and nitrite secretion or collagen and laminin production. However, the metabolic activity was slightly decreased (pâ=â0.0374) and the membrane integrity marginally impaired (pâ=â0.0404) in cells from older donors. Two out of three senescence assays detected more senescence markers in cells from older donors.According to the assays applied here, HAEC from young and elderly donors up to the age of 63 years could be judged equally suitable for autologous cellularization strategies. However, this finding should be regarded with caution due to the extremely large variability between individual donors. Further studies comprising a larger sample size are necessary to investigate this issue more thoroughly.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Adolescente , Adulto , Fatores Etários , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Cyclophosphamide (CPA) is one of the most successful anticancer prodrugs that becomes effective after biotransformation in the liver resulting in the toxic metabolite acrolein. Cancer is often accompanied by thromboembolic events, which might be a result of dysfunctional endothelial cells due to CPA treatment. Here, the effect of 1â¯mM CPA or acrolein (10/50/100/500⯵M) on human umbilical vein endothelial cells (HUVECs) was analyzed after two days of treatment. The addition of CPA or 10⯵M acrolein did not affect HUVECs. However, concentrations of 100⯵M and 500⯵M acrolein significantly reduced the number of adherent cells by 86⯱â¯13% and 99⯱â¯1% and cell viability by 51⯱â¯29% and 93⯱â¯8% compared to the control. Moreover, pronounced stress fibers as well as multiple nuclei were observed and von Willebrand factor (vWF) was completely released. Lactate dehydrogenase was 8.5⯱â¯7.0-fold and 252.9⯱â¯42.9-fold increased showing a loss of cell membrane integrity. The prostacyclin and thromboxane secretion was significantly increased by the addition of 500⯵M acrolein (43.1⯱â¯17.6-fold and 246.4⯱â¯106.3-fold) indicating cell activation/pertubation. High doses of acrolein led to HUVEC death and loss of vWF production. This effect might be associated with the increased incidence of thromboembolic events in cancer patients treated with high doses of CPA.
Assuntos
Acroleína/toxicidade , Antineoplásicos Alquilantes/toxicidade , Ciclofosfamida/toxicidade , Células Endoteliais/efeitos dos fármacos , Pró-Fármacos/toxicidade , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Epoprostenol/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Cultura Primária de Células , Tromboxanos/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Thrombotic events result from different pathologies and are the underlying causes of severe diseases like stroke or myocardial infarction. Recent basic research now revealed a link between food uptake, food conversion and gut metabolism. Gut microbial production of trimethylamine N-oxide (TMAO) from dietary nutrients like choline, lecithin and L-carnitine was associated with the development of cardiovascular diseases. Within this review we give a systematic overview about the influence of TMAO on blood components like platelets and endothelial cells which both are involved as key players in thrombotic processes. In summary, a mechanistic correlation between the gut microbiome, TMAO and cardiovascular diseases becomes obvious and emphasizes to the significance of the intestinal microbiome.
Assuntos
Plaquetas/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Metilaminas/química , Animais , Humanos , CamundongosRESUMO
BACKGROUND: In most clinical studies older people are underrepresented compared to the demographic reality. However, risk for some severe diseases like cancer typically increase with age. Most insight into cancer treatment comes from mixed-age patient cohorts, leading to a lack of detailed understanding of cancer drug effects in the elderly population. There is growing evidence that cancer drug effects can be influenced by dehydration conditions often found in older people. Colon cancer remains the second leading cause of death by cancer in Europe. Inter- and intra-heterogeneity of tumors contribute to why some individuals do not respond to specific cancer therapies or may often suffer a relapse. OBJECTIVE: Our study applies an in vitro drug test system for simulating treatment with cytostatics of colorectal cancer in elderly patients with dehydration condition. METHODS: Two well-known colon cancer cell lines, Caco-2 and RKO, harboring defined cancer-related mutations, were step-wisely adapted from routine culture medium to a severe hyperosmotic condition (397âmOmol/kg) by adding sodium chloride to the medium. We investigated the effects of these cell culture conditions, which should mimic cellular dehydration in elderly people, on the growth characteristics of the cells. Therefore, cell proliferation was investigated by measuring population doubling times. Furthermore, we investigated how the metabolic activity of the cells was influenced by treatment with different concentrations of cyclophosphamide (CPA) under normal and hyperosmotic conditions. RESULTS: We found that Caco-2 and RKO cell lines have an identical cell doubling time of 23 hours in normosmotic medium. However, hyperosmotic medium lifted the doubling time of Caco-2 cells to 31 hours while that of RKO cells did not change. Despite reduced cell proliferation rates, hyperosmotic medium sensitized Caco-2 cells to treatment with 10âmM CPA for 48 hours as measured by metabolic activity assays on ATP levels. CONCLUSIONS: The two investigated colon cancer cells lines reacted differently to hyperosmotic conditions. Only the growth of Caco-2 cells was reduced by increased osmolality. Despite this reduced growth their sensitivity to an alkylating cytostatic agent was even slightly increased. We are now in line to examine these effects in more detail and with more tumor cell lines.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Desidratação/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Células CACO-2 , Células Cultivadas , Humanos , Concentração OsmolarRESUMO
In cancer therapy, a number of drugs with different mechanisms of action are in clinical use, which act directly after administration without metabolism, while others only become active in the metabolites produced in the liver. Such drugs/metabolites - especially when administered parenterally - interact in high concentrations with the endothelium. Whether this induces adverse responses of the endothelial cells (EC) is barely studied for many medicaments.This pilot in vitro study revealed that the addition of cyclophosphamide (CPA) to the culture medium (5 or 10âmM, respectively) showed a clear influence on EC compared to non-treated EC: The number of adherent human vein endothelial cells (HUVEC) decreased by the addition of CPA in a concentration-dependent manner compared to the untreated control, whereby the vitality of adherent cells was not affected. In addition, concomitant with activation of the adherent HUVEC, increased migratory activity occurred.These results are in agreement with clinical events like thromboses in patients in compromised condition under therapy with CPA, as the detachment of EC might induce responses of circulating platelets leading to the adherence and aggregation with the risk of the formation of thrombi. Whether CPA acts directly or via toxic metabolites on EC will be examined in more detail in following studies.
Assuntos
Ciclofosfamida/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Imunossupressores/uso terapêutico , Neoplasias/tratamento farmacológico , Ciclofosfamida/farmacologia , Humanos , Imunossupressores/farmacologiaRESUMO
BACKGROUND: Physical and chemical characteristics of implant materials determine the fate of long-term cardiovascular devices. However, there is still a lack of fundamental understanding of the molecular mechanisms occurring in the material-tissue interphase. In a previous study, soft covalently crosslinked poly(n-butyl acrylate) networks (cPnBA) were introduced as sterilizable, non-toxic and immuno-compatible biomaterials with mechanical properties adjustable to blood vessels. Here we study the influence of different surface treatments in particular oxygen plasma modification and fibrinogen deposition as well as a combinatorial approach on the adhesion and viability of fibroblasts. MATERIAL AND METHODS: Two types of cPnBA networks with Young's moduli of 0.19±0.01 MPa (cPnBA04) and 1.02±0.01 MPa (cPnBA73) were synthesized and post-modified using oxygen plasma treatment (OPT) or fibrinogen coating (FIB) or a combination of both (OPT+FIB). The water contact angles of the differently post-treated cPnBAs were studied to monitor changes in the wettability of the polymer surfaces. Because of the key role of vascular fibroblasts in regeneration processes around implant materials, here we selected L929 fibroblasts as model cell type to explore morphology, viability, metabolic activity, cell membrane integrity as well as characteristics of the focal adhesions and cell cytoskeleton on the cPnBA surfaces. RESULTS: Compared to non-treated cPnBAs the advancing water-contact angles were found to be reduced after all surface modifications (pâ<â0.05, each), while lowest values were observed after the combined surface treatment (OPT+FIB). The latter differed significantly from the single OPT and FIB. The number of adherent fibroblasts and their adherence behavior differed on both pristine cPnBA networks. The fibroblast density on cPnBA04 was 743±434 cells·mm-2, was about 6.5 times higher than on cPnBA73 with 115±73 cells·mm-2. On cPnBA04 about 20% of the cells were visible as very small, round and buckled cells while all other cells were in a migrating status. On cPnBA73, nearly 50% of fibroblasts were visible as very small, round and buckled cells. The surface functionalization either using oxygen plasma treatment or fibrinogen coating led to a significant increase of adherent fibroblasts, particularly the combination of both techniques, for both cPnBA networks. It is noteworthy to mention that the fibrinogen coating overruled the characteristics of the pristine surfaces; here, the fibroblast densities after seeding were identical for both cPnBA networks. Thus, the binding rather depended on the fibrinogen coating than on the substrate characteristics anymore. While the integrity of the fibroblasts membrane was comparable for both polymers, the MTS tests showed a decreased metabolic activity of the fibroblasts on cPnBA. CONCLUSION: The applied surface treatments of cPnBA successfully improved the adhesion of viable fibroblasts. Under resting conditions as well as after shearing the highest fibroblast densities were found on surfaces with combined post-treatment.
Assuntos
Acrilatos/metabolismo , Fibroblastos/metabolismo , Polímeros/metabolismo , Adesão Celular , Sobrevivência Celular , Fibroblastos/citologia , Humanos , Propriedades de SuperfícieRESUMO
Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.
Assuntos
Apoptose , Reparo do DNA , Poli(ADP-Ribose) Polimerases/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular , Dano ao DNA , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Timo/citologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.
Assuntos
Metilnitronitrosoguanidina/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Linhagem Celular Transformada , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Raios gama , Técnicas de Transferência de Genes , Dados de Sequência Molecular , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Polyomavirus/efeitos dos fármacos , Polyomavirus/genética , Polyomavirus/fisiologia , Replicon , Replicação Viral/efeitos dos fármacos , Replicação Viral/efeitos da radiaçãoRESUMO
Poly(ADP-ribose) polymerase (PARP) is an evolutionally conserved nuclear protein present in most eukaryotic species and catalyzes the formation of ADP-ribose polymers covalently attached to proteins. PARP is strongly activated by DNA single- or double-strand breaks and is thought to be involved in cellular responses to DNA damage. Based on the SV40-transformed Chinese hamster cell line CO60, we had established stable transfectants that overexpress the PARP DNA-binding domain conditionally. DNA-binding domain overexpression led to trans-dominant inhibition of poly(ADP-ribosyl)ation and sensitized the cells to genotoxic agents. Using the amplification of chromosomally integrated SV40 DNA as an indicator system, we show here that trans-dominant PARP inhibition potentiates genetic instability induced by N-methyl-N'-nitro-N-nitrosoguanidine treatment of cells.
Assuntos
DNA/metabolismo , Amplificação de Genes , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Células CHO , Carcinógenos/farmacologia , Linhagem Celular Transformada , Cricetinae , DNA/efeitos dos fármacos , Dano ao DNA , Metilnitronitrosoguanidina/farmacologia , Poli Adenosina Difosfato Ribose/metabolismo , Vírus 40 dos Símios , TransfecçãoRESUMO
Poly(ADP-ribose) polymerase (PARP4) catalyzes the formation of ADP-ribose polymers covalently attached to proteins by using NAD+ as substrate. PARP is strongly activated by DNA single- or double-strand breaks and is thought to be involved in cellular responses to DNA damage. We characterized a dominant negative PARP mutant, i.e. the DNA-binding domain of this enzyme, whose overexpression in cells leads to increased genetic instability following DNA damage. In order to study whether PARP activity is also implicated in the process of tumorigenesis, we generated stably transfected HeLa cell clones with constitutive overexpression of dominant negative PARP and investigated tumor formation of these clones in nude mice. We found that inhibition of PARP activity dramatically reduces tumor forming ability of HeLa cells. Moreover, we provide strong evidence that the observed reduction in tumor forming ability is due to increased tumor cell apoptosis in vivo. Viewed together, our data and those from other groups show that inhibition of PARP enzyme activity interferes with DNA base excision repair and leads to increased genetic instability and recombination but, on the other hand, can sensitize cells to apoptotic stimuli and by this mechanism may prevent tumor formation.
Assuntos
Apoptose/genética , Neoplasias Experimentais/patologia , Poli(ADP-Ribose) Polimerases/genética , Animais , Células HeLa , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/enzimologia , Inibidores de Poli(ADP-Ribose) PolimerasesRESUMO
Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics.
Assuntos
Autoanticorpos/imunologia , Neoplasias Laríngeas/imunologia , Miastenia Gravis/imunologia , Receptores Nicotínicos/biossíntese , Células Cultivadas , Humanos , Neoplasias Laríngeas/patologia , Miastenia Gravis/sangueRESUMO
Many cellular enzymes use NAD+ as coenzyme or substrate, depending on the nature of the enzymatic reaction. Under certain conditions the cellular NAD+ concentration may become rate-limiting for such enzymes. For instance, when eucaryotic cells are exposed to high concentrations of DNA-damaging agents, the resulting DNA strand breaks may stimulate the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to such an extent that the cellular pool of NAD+, which is the substrate for this enzyme, is severely depleted, possibly leading to acute cell death. Here we show that NAD+ concentrations in CV-1 monkey and CO60 hamster cells can be raised 3- to 4-fold by electrotransfection of NAD+. This additional NAD+ is indeed available for PARP to synthesize higher-than-normal amounts of poly(ADP-ribose) after treatment with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. NAD+ loading of cells by electrotransfection may be useful also for the study of other cellular reactions in which NAD+ is involved.