Bioconversion of Aflatoxin G1 to Aflatoxin B3 by selected fungi

Aflatoxin G₁ (AFG₁) was transformed into aflatoxin B₃ (AFB₃) by the fungiRhodotorula sp,Sporobolomyces sp,Rhizopus oryzae NRRL395,Pythium ultimum, Aspergillus terreus, A clavatus and Penicillium frequentans grown in a medium containing AFG₁, Difco potato dextrose broth, yeast extract, and peptone both in liquid shaken cultures and in solid static cultures at 25°C in the dark. A maximum rate of transformation of 10 % was obtained after 2 to 3 weeks of incubation. The transformation was correlated with an increase in the pH of the media from 5.7 –5.9 to 8.3 – 8.8.Saccharomyces cerevisiae also transformed AFG₁ into AFB₃, but at a slower rate; the pH of the media did not reach above 8.0 until 5 weeks after incubation. No transformation was observed whenA niger andP chrysogenum were tested; in both cases, no increase in pH was noticed. However, some transformation of AFG₁ to AFB₃ by both fungi was observed when the initial pH of the media was adjusted to 9.0. The rate of transformation increased to 15 – 20% in the static culture where the same medium was adsorbed onto vermiculite andRhizopus andAspergilli gave the highest increase in AFB₃ yield.     

Regenerative potential of human skeletal muscle during aging

In this study, we have investigated the consequences of aging on the regenerative capacity of human skeletal muscle by evaluating two parameters: (i) variation in telomere length which was used to evaluate the in vivo turn-over and (ii) the proportion of satellite cells calculated as compared to the total number of nuclei in a muscle fibre. Two skeletal muscles which have different types of innervation were analysed: the biceps brachii, a limb muscle, and the masseter, a masticatory muscle. The biopsies were obtained from two groups: young adults (23 +/- 1.15 years old) and aged adults (74 +/- 4.25 years old). Our results showed that during adult life, minimum telomere lengths and mean telomere lengths remained stable in the two muscles. The mean number of myonuclei per fibre was lower in the biceps brachii than in the masseter but no significant change was observed in either muscle with increasing age. However, the number of satellite cells, expressed as a proportion of myonuclei, decreased with age in both muscles. Therefore, normal aging of skeletal muscle in vivo is reflected by the number of satellite cells available for regeneration, but not by the mean number of myonuclei per fibre or by telomere lengths. We conclude that a decrease in regenerative capacity with age may be partially explained by a reduced availability of satellite cells.     

Age-related appearance of tubular aggregates in the skeletal muscle of almost all male inbred mice

Tubular aggregates (TAs) which have been recently observed in a few mouse myopathies are identical to those described in human diseases. In this study we show that TAs are also found in the skeletal muscle of almost all normal inbred mice strains. In these inbred strains of mice the presence of TAs is shown to be related to both age and sex. Nine different muscles were stained with the modified Gomori trichrome method to reveal the general morphology of the muscles. Anti-SERCA1 ATPase was used to confirm that the TAs were in fact accumulations of sarcoplasmic reticulum and anti-MyHC IIB to demonstrate that these accumulations were found exclusively in the type IIB muscle fibers. An ultrastructural study confirmed the observations revealed by light microscopy that the TAs were derived from the sarcoplasmic reticulum. TAs were never observed in female inbred mice and were only found in type IIB glycolytic muscle fibers of male inbred mice. Therefore when analyzing the effect of genetic knock out and knock in experiments on the muscle phenotype of transgenic mice one should be aware that the presence of these aggregates is a non-specific phenomenon induced by inbreeding.     

Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.A gradual degenerative loss of skeletal muscle mass and function is one of the most consistent hallmarks of normal aging. When it reaches defined thresholds, this condition is referred to as sarcopenia (1, 2), and can be associated with disability, poor quality of life, frailty, and increased mortality (3). Aging impacts the morphology, function and biochemical properties of skeletal muscle, but the mechanisms leading to the changes in muscle tissue remain unclear.Proteomics links the muscle functional changes with the protein expression pattern. Several proteomic approaches have already been used to study sarcopenia. Protein profiling of whole tissue homogenates has been performed using two-dimensional gel electrophoresis (2DGE)¹ and mass spectrometry to identify the proteins differentially expressed during aging in rat (4–6) and human muscle (7, 8). Other studies have focused on specific fractions such as mitochondrial proteins (9), phosphoproteins (10), glycoproteins (11), basic proteins (12), or calpain interacting proteins (13). The few proteomic studies available on human skeletal muscle are mostly based on the 2DGE approach, which implies focusing on a specific pH range (7, 8). Despite its power of high-resolution, 2DGE presents a limited dynamic range and scarcely resolves low abundance regulatory proteins, hydrophobic proteins, and proteins with extreme pI and/or M_r (14).To circumvent these limitations, we propose in the present study to apply a label-free protein profiling based on a shotgun proteomics approach. This technique permits to identify proteins in a complex mixture after trypsin hydrolysis, using a combination of high performance liquid chromatography and mass spectrometry. In a shotgun analysis previously performed on whole muscle extracts, the major isoforms of myosin heavy-chain comprise ∼42% of the total spectra (15). Because these major isoforms may hamper identification of other proteins, we decided to precipitate myofibrils at low ionic strength (16, 17) and to focus on the soluble fraction. In this paper, we present the analytical steps of label-free quantitation, which resulted in the identification and quantitation of 255 muscle proteins common to all ten individuals. The comparison of protein profiling between mature and older women highlighted 35 differentially expressed proteins during aging, 25 proteins that have not previously been related to muscle aging. The functional interactions network linking these proteins showed that the two main biological processes were represented by proteins involved in energy metabolism and contractile proteins.     

Differences in the Expression and Distribution of Flotillin-2 in Chick,Mice and Human Muscle Cells

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.     

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.