The Fusarium oxysporum gnt2, Encoding a Putative NAcetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed colocalization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity

Mitochondrial dynamics in hepatocytes from mice fed caloric restriction diets with different fat sources

Resumen del trabajo presentado al XIV Congresos de la Sociedad Española de Biología Celular, celebrado en Málaga del 12 al 15 de diciembre de 2011.Peer reviewe

Mitochondrial ultrastructure and markers of dynamics in hepatocytes from aged, calorie restricted mice fed with different dietary fats

PMCID: PMC4104696In this paper we analyzed changes in hepatocyte mitochondrial mass and ultrastructure as well as in mitochondrial markers of fission/fusion and biogenesis in mice subjected to 40% calorie restriction (CR) for 18. months versus ad libitum-fed controls. Animals subjected to CR were separated into three groups with different dietary fats: soybean oil (also in controls), fish oil and lard. Therefore, the effect of the dietary fat under CR was studied as well. Our results show that CR induced changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. Also, mean number of mitochondrial cristae and lengths were significantly higher in all CR groups compared with controls. Finally, CR had no remarkable effects on the expression levels of fission and fusion protein markers. However, considerable differences in many of these parameters were found when comparing the CR groups, supporting the idea that dietary fat plays a relevant role in the modulation of CR effects in aged mice.Supported by NIH grant 1R01AG028125-01A1 (to JJR, PN and JMV), Ministerio de Economía y Competitividad and European FEDERBFU2011-23578 (to JMV), Junta de Andalucía Proyectos de Excelencia grant P09-CVI-4887 (to JMV), Junta de Andalucía Proyectos Internacionales (to JMV), and BIO-276 (Junta de Andalucía and the University of Córdoba, to JMV and EGC). JALD and LFdR were funded by predoctoral fellowships of the Spanish Ministerio de Educación and by BIO-276. HK was funded by a predoctoral fellowship of the Agencia Española de Cooperación Internacional al Desarrollo and by BIO-276.Peer Reviewe

The influence of dietary fat source on liver and skeletal muscle mitochondrial modifications and lifespan changes in calorie-restricted mice

The Membrane Theory of Aging proposes that lifespan is inversely related to the level of unsaturation in membrane phospholipids. Calorie restriction (CR) without malnutrition extends lifespan in many model organisms, which may be related to alterations in membrane phospholipids fatty acids. During the last few years our research focused on studying how altering the predominant fat source affects the outcome of CR in mice. We have established four dietary groups: one control group fed 95 % of a pre-determined ad libitum intake (in order to prevent obesity), and three CR groups fed 40 % less than ad libitum intake. Lipid source for the control and one of the CR groups was soybean oil (high in n-6 PUFA) whereas the two remaining CR groups were fed diets containing fish oil (high in n-3 PUFA), or lard (high in saturated and monounsaturated fatty acids). Dietary intervention periods ranged from 1 to 18 months. We performed a longitudinal lifespan study and a cross-sectional study set up to evaluate several mitochondrial parameters which included fatty acid composition, H+ leak, activities of electron transport chain enzymes, ROS generation, lipid peroxidation, mitochondrial ultrastructure, and mitochondrial apoptotic signaling in liver and skeletal muscle. These approaches applied to different cohorts of mice have independently indicated that lard as a fat source often maximizes the effects of 40 % CR on mice. These effects could be due to significant increases of monounsaturated fatty acids levels, in accordance with the Membrane Theory of Aging.Supported by NIH grant 1R01AG028125 (to JJR, PN and JMV), Ministerio de Economía y Competitividad BFU2011-23578 (to JMV), Junta de Andalucía Proyectos de Excelencia grant P09-CVI-4887 (to JMV), Junta de Andalucía Proyectos Internacionales grant (to JMV), and BIO-276 (Junta de Andalucía and the University of Córdoba, to JMV). RdC is funded by the Intramural Research program of the NIA/NIH. JALD, JA, LFdR and EGC were funded by a predoctoral fellowship of the Spanish Ministerio de Educación and BIO-276. HK was funded by a predoctoral fellowship of the Agencia Española de Cooperación Internacional al Desarrollo and BIO-276. MCR and MdR were supported by BIO-276.Peer Reviewe

Conserved and species-specific molecular denominators in mammalian skeletal muscle aging

Aging is a complex phenomenon involving functional decline in multiple physiological systems. We undertook a comparative analysis of skeletal muscle from four different species, i.e. mice, rats, rhesus monkeys, and humans, at three different representative stages during their lifespan (young, middle, and old) to identify pathways that modulate function and healthspan. Gene expression profiling and computational analysis revealed that pathway complexity increases from mice to humans, and as mammals age, there is predominantly an upregulation of pathways in all species. Two downregulated pathways, the electron transport chain and oxidative phosphorylation, were common among all four species in response to aging. Quantitative PCR, biochemical analysis, mitochondrial DNA measurements, and electron microscopy revealed a conserved age-dependent decrease in mitochondrial content, and a reduction in oxidative phosphorylation complexes in monkeys and humans. Western blot analysis of key proteins in mitochondrial biogenesis discovered that (i) an imbalance toward mitochondrial fusion occurs in aged skeletal muscle and (ii) mitophagy is not overtly affected, presumably leading to the observed accumulation of abnormally large, damaged mitochondria with age. Select transcript expression analysis uncovered that the skeletal inflammatory profile differentially increases with age, but is most pronounced in humans, while increased oxidative stress (as assessed by protein carbonyl adducts and 4-hydroxynonenal) is common among all species. Expression studies also found that there is unique dysregulation of the nutrient sensing pathways among the different species with age. The identification of conserved pathways indicates common molecular mechanisms intrinsic to health and lifespan, whereas the recognition of species-specific pathways emphasizes the importance of human studies for devising optimal therapeutic modalities to slow the aging process

SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass

Increased expression of SIRT1 extends the lifespan of lower organisms and delays the onset of age-related diseases in mammals. Here, we show that SRT2104, a synthetic small molecule activator of SIRT1, extends both mean and maximal lifespan of mice fed a standard diet. This is accompanied by improvements in health, including enhanced motor coordination, performance, bone mineral density, and insulin sensitivity associated with higher mitochondrial content and decreased inflammation. Short-term SRT2104 treatment preserves bone and muscle mass in an experimental model of atrophy. These results demonstrate it is possible to design a small molecule that can slow aging and delay multiple age-related diseases in mammals, supporting the therapeutic potential of SIRT1 activators in humans

Adaptación metabólica y estructural de los tejidos mitóticos y postmitóticos a condiciones de retricción calórica en el ratón. Efecto del tipo de grasa en la dieta

In this work it is analyzed the effect of aging and its possible prevention by calorie restriction (CR) in liver and skeletal muscle from mice. These organs were selected as models of mitotic and post-mitotic tissues, respectively. Furthermore, the effect of dietary fat under CR was also investigated. For this purpose, animals submitted to CR were separated into three CR groups with soybean oil (high in ω−6 polyunsaturated fatty acids), fish oil (with a high content of polyunsaturated ω-3 fatty acids) and lard (rich in saturated and monounsaturated fatty acids). Soybean oil was the fat source in control ad libitum fed animals. After 6 and 18 months of CR, animals were killed and different morphological and functional parameters of both organs were studied. In liver, our results show that CR induced changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. Also, mean number of mitochondrial cristae and lengths were significantly higher in all CR groups compared with controls. CR had no remarkable effects on the expression levels of fission (Fis1 and Drp1) and fusion (Mfn1, Mfn2 and OPA1) protein markers after 18 months of CR. However, considerable differences in many of these parameters were found when comparing the CR groups. On the other hand, aging, CR and fat source are all factors that may influence apoptotic signaling in liver. For this reason the combined effect of these factors on a number of apoptosis regulators and effectors were studied as well. An age-linked increase in the mitochondrial apoptotic pathway was detected with CR, including a decrease in Bcl-2/Bax ratio, an enhanced release of cytochrome c to the cytosol and higher caspase-9 activity. Apoptotic index (DNA fragmentation) and mean nuclear area were increased in aged animals with the exception of calorie-restricted mice fed a lard-based fat source. In skeletal muscle, 6 and 18 months of CR partially prevented the decreased size of red and white fibers observed in aged control mice. At the ultrastructural level, mean area of myofibrils and planimetric parameters of subsarcolemmal and intermyofibrilar mitochondria also changed during aging and in the different groups of CR and similar results were obtained when the expression levels of protein related to mitochondrial fission, fusion and function (Sirt3 and VDAC) were analyzed...En este trabajo se estudia el efecto del envejecimiento y su posible prevención mediante restricción calórica (RC) en hígado y músculo esquelético de ratón. Dichos órganos fueron elegidos como modelo de tejidos mitótico y postmitótico respectivamente. Asímismo se investigó el efecto de la grasa de la dieta. Para ello, los animales sometidos a RC se separaron en tres grupos que fueron alimentados con diferentes fuentes de grasa: aceite de soja (rico en ácidos grasos poliinsaturados tipo ω−6), aceite de pescado (rico en en ácidos grasos poliinsaturados tipo ω−3) y manteca (rica en ácidos grasos saturados y monoinsaturados). La fuente de grasa en los animals control alimentados ad libitum fue aceite de soja. Tras 6 y 18 meses en dichas condiciones, los animales fueron sacrificados y se analizaron diversos parámetros morfológicos y funcionales de ambos órganos. En hígado, los resultados mostraron que la RC indujo cambios en el tamaño de hepatocitos y las mitocondrias así como en el número y la fracción de volumen ocupado por mitocondrias en el hepatocito. De igual forma, en número medio de crestas mitocondriales y su longitud aumentaron de forma considerable en todos los grupos de RC en comparación con los animales control. La RC no tuve efectos considerables en los niveles de expresión de proteínas relacionadas con la fisión (Fis1 y Drp1) y fusión mitocondrial (Mfn1, Mfn2 y OPA1) tras 18 meses de RC. Sin embargo, éstas aparecieron al comparar entre sí los distintos grupos de RC. Por otra parte, el envejecimiento, la RC y la fuente de grasa son factores que pueden influir en la señalización apoptótica en hígado. Por dicha razón se estudió el efecto combinado de esos factores sobre diversos reguladores y efectores de la apoptosis, encontrándose un incremento dependiente de la edad en la ruta mitocondrial apoptótica en los animales sometidos a RC entre los que se incluyen disminución de la relación Bcl-2/Bax, aumento de la liberación de citocromo c al citosol e incremento en la actividad caspasa-9. El índice apoptótico (fragmentación de DNA) y el área media nuclear aumentaron en los animales más envejecidos con la excepción de los que fueron alimentados con manteca y en condiciones de RC. En músculo esquelético, 6 y 18 meses de RC previnieron en parte la disminución del tamaño de fibras rojas y blancas que se había observado en los ratones control. A nivel ultraestructural, el área media de las fibrillas y los..

Knowledge, Awareness, and Practices toward Colorectal Cancer and Its Dietary and Lifestyle-Related Risk Factors among Jordanian University Students: A Cross-Sectional Study

Background. Globally, colorectal cancer (CRC) incidence is rising, and it is a leading cause of mortality, with greater death rates pronounced in developing countries, including Jordan. Understanding knowledge and awareness of etiologic factors, unhealthy lifestyles, and dietary patterns is crucial for combating ailments. Hence, this study is aimed at investigating the level of knowledge and awareness of CRC-related risk factors, practices, and possible associations of studied variables among young Jordanians. Methodology. A cross-sectional, observational study was conducted using an online self-reported assessment of anthropometrics, knowledge, awareness, and dietary and lifestyle practices toward CRC and its related risk factors. Results. A study of 795 Jordanian university students found that 93.8% were Jordanians, 73.0% were female, aged 18-24, and single. Most participants were from medical and science schools (69.4%). The vast majority (about 84%) were found to have good knowledge and awareness of CRC and its risk factors, but this was not reflected in their dietary practices. There are significant differences in physical activity, smoking, vegetable consumption, and serving sizes of red meat and processed meats between the sexes. Academic study specialties significantly impact knowledge and awareness. Conclusion. The study reveals that while young Jordanian university students have good knowledge and awareness about CRC and its risk factors, these levels are not reflected in their dietary behaviors and food choices for CRC prevention, highlighting the need for national programs to improve these practices, particularly in the younger population