Long term betaine supplementation regulates genes involved in lipid and cholesterol metabolism of two muscles from an obese pig breed.

This study evaluates the effects of betaine supplementation (1gkg-1 for 20weeks) on the regulation of genes involved in lipid and cholesterol metabolism of Longissimus lumborum and Biceps femoris from obese Alentejano pigs. Betaine supplementation led to an increase in total cholesterol in both muscles, complementing results previously published indicating a significant increase on the intramuscular lipid content. The expression of twelve genes involved in lipogenesis, lipolysis/FA oxidation, FA transport, and cholesterol metabolism, as well as two transcription factors were also evaluated. Genes related to lipid and cholesterol synthesis plus FA transport were consistently up-regulated in both muscles of betaine fed pigs. On the other hand, genes related to lipolysis/FA oxidation were not affected or down-regulated by betaine supplementation. Our data suggest that the underlying mechanism regulating IMF and cholesterol accumulation in Alentejano pigs supplemented with betaine is associated with the up-regulation of genes involved in lipid synthesis, FA transport, and cholesterol synthesis.

Oxidative Stress, DNA, Cell Cycle/Cell Cycle Associated Proteins and Multidrug Resistance Proteins: Targets of Human Amniotic Membrane in Hepatocellular Carcinoma.

The anticancer effects of human amniotic membrane (hAM) have been studied over the last decade. However, the action mechanisms responsible for these effects are not fully understood until now. Previously results reported by our team proved that hAM is able to induce cytotoxicity and cell death in hepatocellular carcinoma (HCC), a worldwide high incident and mortal cancer. Therefore, this experimental study aimed to investigate the cellular targets of hAM protein extracts (hAMPE) in HCC through in vitro studies. Our results showed that hAMPE is able to modify oxidative stress environment in all HCC cell lines, as well as its cell cycle. hAMPE differently targets deoxyribonucleic acid (DNA), P21, P53, ß-catenin and multidrug resistance (MDR) proteins in HCC cell lines. In conclusion, hAMPE has several targets in HCC, being clear that the success of this treatment depends of a personalized therapy based on the biological and genetic characteristics of the tumor.

Selective cytotoxicity and cell death induced by human amniotic membrane in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a worldwide high incidence and mortality. For this reason, it is essential to invest in new therapies for this type of cancer. Our team already proved that human amniotic membrane (hAM) is able to inhibit the metabolic activity of several human cancer cell lines, including HCC cell lines. Taking into account the previously performed work, this experimental study aimed to investigate the pathways by which hAM protein extracts (hAMPEs) act on HCC. Our results showed that hAMPE reduce the metabolic activity, protein content and DNA content in a dose- and time-dependent manner in all HCC cell lines. This therapy presents selective cytotoxicity, since it was not able to inhibit a non-tumorigenic human cell line. In addition, hAMPE induced cell morphology alterations in all HCC cell lines, but death type is cell line dependent, as proved by in vitro and in vivo studies. In conclusion, hAMPE have a promising role in HCC therapy, since it is capable of inducing HCC cytotoxicity and cell death.

Effect of amniotic membrane proteins in human cancer cell lines: an exploratory study.

Human amniotic membrane (hAM) has recently drawn attention as an upcoming anti-cancer therapy. Regarding the strategies which have already investigated, little is known about hAM protein extracts (hAMPE) effect on cancer. So, this work aims to study the effect of hAMPE in metabolic activity of several human cancer cell lines. hAMPE were mechanically obtained, thus avoiding the effect of detergents and other reagents commonly used in protein extraction under the cell lines studied. After quantification of proteins in hAMPE, their effect on the metabolic activity of 21 human cancer cell lines was assessed by 3-(4,5-dimethylthia-zolyl-2)2,5-diphenyltetrazolium bromide (MTT) assay. Our results indicate that there is an inhibition of metabolic activity until 25 and 50% in two and seven cell lines, respectively. Five cell lines proved to be very sensitive to hAMPE, being its metabolic activity more than 50% inhibited. Our results show that hAMPE can inhibit the metabolic activity of some human cancer cell lines. However, research about this cell line-dependent response to hAMPE becomes indispensable.

A survey of entomopathogenic nematode species in continental Portugal.

Entomopathogenic nematodes (EPN) are lethal parasites of insects, used as biocontrol agents. The objectives of this work were to survey the presence of EPN in continental Portugal and to characterize the different species. Of the 791 soil samples collected throughout continental Portugal, 53 were positive for EPN. Steinernema feltiae and Heterorhabditis bacteriophora were the two most abundant species. Analysis of EPN geographical distribution revealed an association between nematode species and vegetation type. Heterorhabditis bacteriophora was mostly found in the Alentejo region while S. feltiae was present in land occupied by agriculture with natural vegetation, broadleaved forest, mixed forest and transitional woodland-shrub, agro-forestry areas, complex cultivated patterns and non-irrigated arable land. Although no clear association was found between species and soil type, S. feltiae was typically recovered from cambisols and H. bacteriophora was more abundant in lithosols. Sequencing of the internal transcribed spacer (ITS) region indicated that S. feltiae was the most abundant species, followed by H. bacteriophora. Steinernema intermedium and S. kraussei were each isolated from one site and Steinernema sp. from two sites. Phylogenetic analyses of ITS, D2D3 expansion region of the 28S rRNA gene, as well as mitochondrial cytochrome c oxidase subunit I (COXI) and cytochrome b (cytb) genes, was performed to evaluate the genetic diversity of S. feltiae and H. bacteriophora. No significant genetic diversity was found among H. bacteriophora isolates. However, COXI seems to be the best marker to study genetic diversity of S. feltiae. This survey contributes to the understanding of EPN distribution in Europe.

Molecular characterization of Portuguese populations of the pinewood nematode Bursaphelenchus xylophilus using cytochrome b and cellulase genes.

Bursaphelenchus xylophilus is the causal agent of pine wilt disease and a worldwide pest with high economic impact. Since its first diagnosis in Portugal in 1999, it has been subjected to quarantine measures with impact on forest health and ecosystem stability, significantly affecting international trade of wood products. The disease was detected in the north and centre of continental Portugal and, since 2008, the whole country has been considered an affected area. Recently, it was detected in Madeira Island. In order to avoid new outbreaks, it has become of major importance to understand the patterns of spread, introduction points and to characterize the new populations from continental Portugal and Madeira Island. Mitochondrial cytochrome b (cytb) and parasitic cellulase gene sequences were used to evaluate the genetic relationships among isolates that could indicate possible origins of the new outbreaks. Portuguese isolates were compared with isolates from USA, China, Japan and South Korea, in order to investigate possible infection pathways and disease spread patterns in Portugal. Phylogenetic trees based on both genes show that Portuguese isolates group with Asian isolates. Isolates from USA are in a separate position in both gene trees. However, the phylogenetic tree based on the cellulase gene sequences shows higher differentiation among Portuguese isolates than that of cytb. These results agree with those previously obtained using inter-simple sequence repeats (ISSR). This was the first study to use cytb and cellulase genes to characterize pinewood nematode (PWN) populations. This study suggests that cellulase is a better marker than cytb to study genetic diversity in B. xylophilus.

Amniotic membrane: from structure and functions to clinical applications.

Amniotic membrane (AM) or amnion is a thin membrane on the inner side of the fetal placenta; it completely surrounds the embryo and delimits the amniotic cavity, which is filled by amniotic liquid. In recent years, the structure and function of the amnion have been investigated, particularly the pluripotent properties of AM cells, which are an attractive source for tissue transplantation. AM has anti-inflammatory, anti-bacterial, anti-viral and immunological characteristics, as well as anti-angiogenic and pro-apoptotic features. AM is a promoter of epithelialization and is a non-tumorigenic tissue and its use has no ethical problems. Because of its attractive properties, AM has been applied in several surgical procedures related to ocular surface reconstruction and the genito-urinary tract, skin, head and neck, among others. So far, the best known and most auspicious applications of AM are ocular surface reconstruction, skin applications and tissue engineering. However, AM can also be applied in oncology. In this area, AM can prevent the delivery of nutrients and oxygen to cancer cells and consequently interfere with tumour angiogenesis, growth and metastasis.

Tumour hypoxia and technetium tracers: in vivo studies.

INTRODUCTION: Hypoxia is a biochemical condition where reduced oxygen partial pressure at tissue level occurs. This metabolic situation can lead to resistance to radio and chemotherapy. In malignant solid tumours, hypoxia is a common characteristic, having a great impact at biological level, being of tremendous importance for complete understanding of tumour progression. OBJECTIVES: We studied the behavior of 99mTc-HL91 in vivo, using an animal model based on Balb-c nu/nu mice with a xenotransplant of the human colorectal adenocarcinoma cell line, WiDr. MATERIAL AND METHODS: In vivo studies using an animal model of xenograft on Balb/c nu/nu nude mice were carried out. This model, allowed us to evaluate the radiopharmaceutical biodistribution and to calculate tumour/muscle ratio, acquired after 99mTc-HL91 injection. We also performed ex vivo studies, using the excised tumours to access viability and to characterize the intracellular production of reactive oxygen species and the status of mitochondrial membrane potential through flow cytometry. RESULTS AND DISCUSSION: The biodistribution after 99mTc-HL91 injection showed urinary and hepatobilliary excretion in similar proportions and tumour uptake around 4.4% of administered activity. This uptake was higher at the bigger tumours. Through flow cytometry we observed that larger tumours have a higher amount of reactive oxygen species and a decrease in mitochondrial membrane potential. CONCLUSIONS: 99mTc-HL91 allowed a non-invasive evaluation of the solid tumours oxidative state by nuclear medicine functional imaging. This information can be of high importance at the pre-treatment estimation of this type of tumours.

Halogen atom effect on photophysical and photodynamic characteristics of derivatives of 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin.

Brominated and iodinated derivatives of 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin were synthesised directly from the corresponding aldehydes. Photophysical and photochemical properties, singlet oxygen formation quantum yields, photobleaching and log P were measured. Cellular uptake measurements and cytotoxicity assays on WiDr and A375 tumour cell lines were performed. 5,10,15,20-Tetrakis(2-bromo-5-hydroxyphenyl)porphyrin showed the best cytotoxicity with values of IC(50) of 113 nM over WiDr cells and 52nM over A375 cells.

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum.

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.

Moderately acidophilic mesorhizobia isolated from chickpea.

AIMS: Our aim was to evaluate the effect of acid and alkaline pH on chickpea rhizobia, and on chickpea-rhizobia symbiosis. METHODS AND RESULTS: Forty-seven rhizobia isolates obtained from 12 Portuguese soils were grown at pH 5, 7 and 9. Among these, 26 grew more at pH 5 than at 7, suggesting the existence of acidophiles. All isolates were identified as mesorhizobia by 16S rDNA partial sequence analysis. Molecular phylogeny of isolates based on partial 16S rDNA sequences suggests that pH tolerance might be species related. Further studies were conducted with six isolates, which were able to grow at acid pH. Isolates PT-35 and 64b grow optimally at pH 6-5-7, with a minimal pH range from 5 to 3, and may thus be considered as moderately acidophilic. Both isolates belong to a previously identified putative new Mesorhizobium species, based on 16S rDNA sequence. CONCLUSIONS: Two moderately acidophilic mesorhizobia isolated from chickpea were identified (PT-35 and 64b). A positive correlation was found between the symbiotic effectiveness at low pH and the acid tolerance of rhizobia isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on moderately acidophilic mesorhizobia, and is an important contribution for the development of highly effective inoculants for chickpea in acid soils.

Comparison of chickpea rhizobia isolates from diverse Portuguese natural populations based on symbiotic effectiveness and DNA fingerprint.

AIMS: To test the hypothesis that differences in chickpea yields obtained in four distinct Portuguese regions (Beja, Elvas-Casas Velhas, Elvas-Estação Nacional de Melhoramento de Plantas (ENMP) and Evora) could be due to variation between the natural rhizobia populations. METHODS AND RESULTS: Estimation of the size of the different rhizobial populations showed that Elvas-ENMP population was the largest one. Elvas-ENMP population also revealed a higher proportion of isolates carrying more than one plasmid. Assessment of genetic diversity of the native rhizobia populations by a DNA fingerprinting PCR method, here designated as DAPD (Direct Amplified Polymorphic DNA), showed a higher degree of variation in Elvas-ENMP and Beja populations. The symbiotic effectiveness (SE) of 39 isolates was determined and ranged 13-34%. Statistical analysis showed that SE was negatively correlated with plasmid number of the isolate. CONCLUSIONS: The largest indigenous rhizobia population was found in Elvas-ENMP. DAPD pattern and plasmid profile analysis both suggested a higher genetic diversity among the populations of Elvas-ENMP and Beja. No relationship was found between SE of the isolates and their origin site. SIGNIFICANCE AND IMPACT OF STUDY: The large native population, rather than the symbiotic performance of individual rhizobia, could contribute to the higher chickpea yields obtained in Elvas-ENMP.

Rhizobia of chickpea from southern Portugal: symbiotic efficiency and genetic diversity.

AIMS: In order to evaluate differences between chickpea rhizobial populations from three geographical areas in southern Portugal (Beja, Elvas and Evora), isolates from the three regions were obtained and analysed. METHODS AND RESULTS: The genetic characterization of the isolates was done by plasmid profiles and restriction analysis of the nifH gene. Symbiotic efficiency of the isolates was also determined. Relationships between geographical origin, symbiotic efficiency and molecular characteristics were established. Beja soil revealed a larger rhizobia population as well as the presence of some of the isolates with higher symbiotic efficiency values. Isolates with a single plasmid showed a significantly higher symbiotic efficiency. CONCLUSION: Genetic and phenotypic differences were detected between the natural rhizobial populations from the three locations. SIGNIFICANCE AND IMPACT OF THE STUDY: The different yield potential with cultivars of chickpea usually obtained in the three regions of southern Portugal could be due to their different natural rhizobial populations.