Probing non-enzymatic glycation of type I collagen: A novel approach using Raman and infrared biophotonic methods

Background

Non-enzymatic glycation is the main post-translational modification of long-life proteins observed during aging and physiopathological processes such as diabetes and atherosclerosis. Type I collagen, the major component in matrices and tissues, represents a key target of this spontaneous reaction which leads to changes in collagen biomechanical properties and by this way to tissue damages.

Methods

The current study was performed on in vitro glycated type I collagens using vibrational microspectroscopies, FT-IR and Raman, to highlight spectral features related to glycation effect.

Results and conclusions

We report a conservation of the triple-helical structure of type I collagen and noticeable variations in the exposure of proline upon glycation. Our data also show that the carbohydrate band can be a good spectroscopic marker of the glycation level, correlating well with the fluorescent AGEs formation with sugar addition.

General significance

These non-invasive and label-free methods can shed new light on the spectral features of glycated collagens and represent an effective tool to study changes in the extracellular matrix observed in vivo during aging or on the advent of a pathological situation.     

Association and in Silico Assignment of Sequences from Turkey BACs

Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.     

Glycated Proteins Can Enhance Photooxidative Stress in Aged and Diabetic Lenses

This study intends to clarify the ability of different carbonyl-containing lens metabolites to form advanced glycation end products, which possess photosensitizer activity and to investigate whether these modified proteins could be implicated in lens photodamage. Calf lens protein was experimentally glycated with either methylglyoxal, glyoxal, ascorbic acid, or fructose to obtain models of aged and diabetic cataractous lenses. Being exposed to 200 J/cm 2 UVA radiation the model glycated proteins produced 2-3-fold more singlet oxygen compared to the unmodified protein and the superoxide radical formation was 30-80% higher than by the native protein. Ascorbylated proteins demonstrated the highest photosensitizer activity. Biological responses of glycation-related photosensitizers were studied on cultured lens epithelial cells irradiated with 40 J/cm 2 UVA. Tissue culture studies revealed a significant increase in thiobarbituric acid reactive substances in the culture medium of lens epithelial cells after irradiation and treatment with glycated proteins. Lens proteins had a protective effect against UVA induced cytotoxicity, however, this protective effect decreased with the increasing photosensitizer activity of experimentally glycated proteins. The documented glycation-related photosensitization could explain the accelerated pathogenic changes in human lens at advanced age and under diabetic conditions.     

Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

Alterations in the balance of cytoskeleton as well as energetic proteins are involved in the cardiac remodeling occurring in dilated cardiomyopathy (DCM). We used two-dimensional DIGE proteomics as a discovery approach to identify key molecular changes taking place in a temporally controlled model of DCM triggered by cardiomyocyte-specific serum response factor (SRF) knock-out in mice. We identified muscle creatine kinase (MCK) as the primary down-regulated protein followed by α-actin and α-tropomyosin down-regulation leading to a decrease of polymerized F-actin. The early response to these defects was an increase in the amount of desmin intermediate filaments and phosphorylation of the αB-crystallin chaperone. We found that αB-crystallin and desmin progressively lose their striated pattern and accumulate at the intercalated disk and the sarcolemma, respectively. We further show that desmin is a preferential target of advanced glycation end products (AGE) in mouse and human DCM. Inhibition of CK in cultured cardiomyocytes is sufficient to recapitulate both the actin depolymerization defect and the modification of desmin by AGE. Treatment with either cytochalasin D or glyoxal, a cellular AGE, indicated that both actin depolymerization and AGE contribute to desmin disorganization. Heat shock-induced phosphorylation of αB-crystallin provides a transient protection of desmin against glyoxal in a p38 MAPK-dependent manner. Our results show that the strong down-regulation of MCK activity contributes to F-actin instability and induces post-translational modification of αB-crystallin and desmin. Our results suggest that AGE may play an important role in DCM because they alter the organization of desmin filaments that normally support stress response and mitochondrial functions in cardiomyocytes.     

Identification and characterization of splicing variants of PLEKHA5 (Plekha5) during brain development

PLEKHA5 (pleckstrin homology domain-containing protein family A, member 5) belongs to the PLEKHA family (PLEKHA1-6); however, the properties of this protein remain poorly characterized. We have identified and characterized two forms of PLEKHA5 mRNA. The long form of PLEKHA5 (L-PLEKHA5) contains 32 exons, encodes 1282 amino acids, and is specifically expressed in the brain; the short form of PLEKHA5 (S-PLEKHA5) is generated by alternative splicing of L-PLEKHA5, contains 26 exons, encodes 1116 amino acids, and is ubiquitously expressed. Both forms of the protein contain putative Trp-Trp (WW) and pleckstrin homology (PH) domains and are located mainly in the cytosol. Developmental and age-dependent expression studies in the mouse brain have shown that Plekha5 is the most abundantly expressed protein at E13.5 with S-Plekha5 dominancy. L-Plekha5 levels increased gradually with the decrease in total Plekha5 levels; moreover, L-Plekha5 became the dominant protein at E17.5, maintaining its dominance throughout adulthood. Protein-lipid overlay assays have indicated that the PH domain of PLEKHA5 specifically interacts with PI3P, PI4P, PI5P, and PI(3,5)P2. These results suggest that the S- to L-conversion of PLEKHA5 (Plekha5) may play an important role in brain development through association with specific phosphoinositides.     

Genomic organization of selected genes in the small monogonont rotifer, Brachionus koreanus

Information of genome structure with its size variation may provide important clues for evolutionary processes at lower taxon level in eukaryotes. Here, we analyzed the compact genome structure of the monogonont rotifer, Brachionus koreanus in the light of transphyletic genome comparison and economic genome usage. To confirm the genome compactness of B. koreanus, we compared the genomic structure of several selected genes with those of human and pufferfish. For example, one of the large genes, DNA-dependent protein kinase (DNA-PK) with dimeric protein Ku70 and Ku80, showed high similarity, even though genomic DNA lengths were quite different. The replication protein As (RPAs) as a heterotrimeric protein also showed a compact genomic structure including all the essential domains and motifs in B. koreanus. Regarding transmembrane protein-containing genes, the B. koreanus P-glycoprotein (P-gp) showed exactly the same topology of the TM domain compared to those of human and pufferfish, even though it had a compact genome structure. In addition, the gene structure of an inducible repair enzyme O(6)-methylguanine DNA methyltransferase (O(6)-MGMT) of B. koreanus showed the highest compactness among the genes tested. The objective of this report is to evaluate the potential for whole genome sequencing and functional genomic research using the monogonont rotifer B. koreanus as a non-model organism that plays important roles in aquatic food-webs. Subsequently, we discussed possible reasons for compact genome structures as well as small and fewer introns from several perspectives. We conclude that the small size genome of B. koreanus would make this species potentially useful for comparative genome structure analysis of non-model species through whole genome sequencing and genetic mapping.     

Alpha-lipoic acid induces apoptosis in hepatoma cells via the PTEN/Akt pathway

We report here that alpha-lipoic acid (alpha-LA), a naturally-occurring antioxidant, scavenges reactive oxygen species (ROS) followed by an increase in apoptosis of human hepatoma cells. Apoptosis induced by alpha-LA was dependent upon the activation of the caspase cascade and the mitochondrial death pathway. alpha-LA induced increases in caspase-9 and caspase-3 but had no significant effect on caspase-8 activity. Apoptosis induced by alpha-LA was found to be mediated through the tensin homologue deleted on chromosome 10 (PTEN)/Akt pathway. Prior to cell apoptosis, PTEN was activated and its downstream target Akt was inhibited. Our findings indicate that increasing ROS scavenging could be a therapeutic strategy to treat cancer.     

Intervention against the Maillard reaction in vivo

The field of Maillard/glycation reactions in vivo has grown enormously during the past 20 years, going from 25 to 500 publications per year. It is now well recognized that many of the “advanced” products form oxidatively or anaerobically and can have deleterious effects on macromolecular and biological function. The feasibility of developing pharmacological agents with beneficial in vivo properties, based on in vitro inhibition of glycation, has been surprisingly successful. This Editorial sets the stage for a series of articles by experts in the field, who have made key contributions to our understanding of the Maillard reaction in vivo.     

Phenology and climate relationships in aspen (Populus tremuloides Michx.) forest and woodland communities of southwestern Colorado

Trembling aspen (Populus tremuloides Michx.) occurs over wide geographical, latitudinal, elevational, and environmental gradients, making it a favorable candidate for a study of phenology and climate relationships. Aspen forests and woodlands provide numerous ecosystem services, such as high primary productivity and biodiversity, retention and storage of environmental variables (precipitation, temperature, snow–water equivalent) that affect the spring and fall phenology of the aspen woodland communities of southwestern Colorado. We assessed the land surface phenology of aspen woodlands using two phenology indices, start of season time (SOST) and end of season time (EOST), from the U.S. Geological Survey (USGS) database of conterminous U.S. phenological indicators over an 11-year time period (2001–2011). These indicators were developed with 250 m resolution remotely sensed data from the Moderate Resolution Imaging Spectroradiometer processed to highlight vegetation response. We compiled data on SOST, EOST, elevation, precipitation, air temperature, and snow water equivalent (SWE) for selected sites having more than 80% cover by aspen woodland communities. In the 11-year time frame of our study, EOST had significant positive correlation with minimum fall temperature and significant negative correlation with fall precipitation. SOST had a significant positive correlation with spring SWE and spring maximum temperature.