Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated

Over the past decade, there have been many reports suggesting the presence of complex carbohydrates on nuclear and cytoplasmic proteins in mammalian cells. Some of the most often cited of these reports deal with the glycosylation of the high mobility group (HMG) proteins. These are relatively abundant chromosomal proteins that are known to be associated with nucleosomes and actively transcribed regions of chromatin. The original report describing HMG protein glycosylation presented several lines of evidence suggesting that these proteins are glycosylated, including carbohydrate compositional analysis and periodic-acid Schiff staining. We have attempted to repeat these observations with more highly purified protein than was utilized in the original study. Using carbohydrate compositional analysis performed by high pH anion exchange chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins. In addition, we found no evidence for the presence of O- GlcNAc, a well known form of nuclear glycosylation. The HMG proteins did react with periodate, suggesting the presence of a modification containing cis-diols on the protein. Several tryptic peptides isolated from HMG 14 and 17 which retained the periodate reactivity had in common lysine residues, suggesting a potential modification of the straightepsilon-amino groups of lysines such as nonenzymatic glycation. Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.      

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic,Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring,SWATH, and MSE

Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified albumin using high resolution accurate mass spectrometry (HR/AM). The glycated peptides were manually inspected and validated for their modification. Further, the fragment ion library was used for quantification of glycated peptides of albumin in the context of diabetes. Targeted Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH) analysis in pooled plasma samples of control, prediabetes, diabetes, and microalbuminuria, has led to identification and quantification of 13 glycated peptides comprised of four AML, seven CML, and two CEL modifications, representing nine lysine sites of albumin. Five lysine sites namely K549, K438, K490, K88, and K375, were observed to be highly sensitive for glycation modification as their respective m/z showed maximum fold change and had both AML and CML modifications. Thus, peptides involving these lysine sites could be potential novel markers to assess the degree of glycation in diabetes.Diabetes is a complex metabolic disorder characterized by prolonged hyperglycemia resulting from defects in insulin secretion, insulin action, or both, leading to abnormalities in carbohydrate, fat, and protein metabolism (1). According to the projection by the International Diabetes Foundation, around 592 million people will be affected by diabetes by the year 2040 (2). Diabetes and its associated complications are becoming global public health problems and posing a serious challenge in disease management. Many studies have implicated advanced glycation end products (AGEs)¹ in the development of insulin resistance, as well as in pathogenesis of diabetic complications (3). The levels of AGEs increase substantially in diabetic plasma due to the hyperglycemic condition. Factors such as oxidative stress, overnutrition, and foods rich in glycating agents promote the formation of AGEs even in nondiabetic condition (4). Oral AGEs foster insulin resistance and diabetes by down-regulation of anti-AGE receptor-1(AGER1), sirtuin 1, and up-regulation of receptor for AGEs (RAGE) (5). AGEs affect glucose uptake, transport and promote insulin resistance in adipocytes (6). While in skeletal muscle cells AGEs inhibit insulin action, mediated through RAGE (7). The AGE-RAGE axis induces oxidative stress, activates proinflammatory pathways and has been considered as a principal pathway in the pathogenesis of diabetes and its complications (8). AGE interacts with RAGE in different cells and tissues, contributing to pathogenesis in diabetes (9). By and large, AGEs contribute to development of insulin resistance leading to diabetes, as well as in the pathogenesis of diabetic complications. Therefore, analysis of plasma AGEs can possibly provide information about the severity of diabetes.Human serum albumin (HSA), one of the most abundant plasma proteins, is highly glycated and contributes predominantly to the plasma AGEs. Apart from its role in pathogenesis, AGE-modified HSA (AGE-HSA) has been suggested as an alternative diagnostic marker to glycated hemoglobin (HbA1c) for monitoring glycemic status in diabetes (10). Although HbA1c is considered the “gold standard” marker, reflecting the glycemic status over the period of 8–10 weeks (1, 10), factors like anemia, blood loss, splenomegaly, and iron deficiency affect HbA1c levels (11). AGE-HSA reflects glycemic status over the preceding 3–4 weeks and has been recommended in gestational diabetes (12). In diabetes, the levels of AGE-HSA increase and were found to be positively correlated with hyperglycemia (13, 14). In addition, several recent studies have suggested that the levels of AGE-HSA are associated with prediabetic condition (15) and microalbuminuria (16). Therefore, quantification of AGE-HSA is of utmost clinical significance. Thus, understanding the site-specific modification and their dynamic transformation to heterogeneous AGEs is quite critical for mass spectrometric quantification.AGEs can be quantified by various approaches, including colorimetric assay, ketoamine oxidase assay, enzyme-linked boronate immunoassay, fluorescence spectroscopy, boronic acid affinity chromatography assay, and mass spectrometry (MS) (17). Among these approaches, MS offers precise characterization of protein glycation, including the amino acid involved in the modification. Most of the AGEs reported in vitro and in vivo were discovered by MS-based techniques (18). AML modification has been extensively studied by different MS approaches. The fragmentation pattern and diagnostic ions for AML rearrangement product has been well established (19, 20). Further specific neutral loss ions of 162 Da, 120 Da, and 84 Da and water loss of 36 Da arising from hexose moiety of glycated peptide were also considered as signature ions to validate the glycation of peptides in HSA (21, 22). Similar characteristic patterns of water loss (18, 36, and 54 Da) ions and immonium ions derived from lysine arising from AML-modified peptide were also used to identify glycated peptides (23, 24). Diagnostic ions serve as the most reliable way of identifying glycated peptide by tandem mass spectrometry. Thus, having a good MS/MS fragment ion is key for precise characterization of glycation. However, the ratio of in vivo AGE-modified to unmodified protein is significantly low, which limits better MS/MS. Therefore, to achieve efficient identification, enrichment of glycated peptides using boronate affinity chromatography (BAC) was adopted prior to MS analysis (25). Further, by using a combination of immunodepletion, enrichment and fractionation strategies, a total of 7,749 unique glycated peptides corresponding to 1,095 native human plasma proteins, 1,592 in vitro glycated human plasma proteins, and 1,664 erythrocyte proteins were identified (26). In these lines, we have previously reported a database search approach for the identification of glycated peptide in a crude or nonenriched sample by untargeted MS/MS or data-independent workflow (27). Glycation is chronic process; a given protein can undergo dynamic heterogeneous transformations as these proteins have varying biological lifespans, influencing the function of a protein. Thus, to assess the degree of glycation at a given pathophysiological condition, precise identification of glycation becomes critical. In this regard, a stable-isotope-dilution tandem mass spectrometry method was employed for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins (28), and ¹³C6-glucose was utilized to quantify glycated proteins in the plasma and erythrocytes (29, 30). In a recent study, the glycation-sensitive peptides of HSA that could serve as markers for early diagnosis of type 2 diabetes were quantified by using an MS-based ¹⁸O-labeling technique (31). However, most of the previous studies have focused on AML modification, rather than other AGE modification. In fact, CML and CEL are the predominant AGEs, constituting up to 80% of total AGEs (32, 33). Diagnostic reporter ions for CML and CEL were reported recently by Prof. Ralf Hoffmann''s group (34). Here, for the first time, we report comprehensive development of an MS/MS fragment ion library for AML, CML, and CEL modifications of albumin. Further, fragment ion library was used as reference for quantification of AML-, CML-, and CEL-modified peptides of albumin in clinical plasma of healthy, prediabetic, diabetic, and microalbuminuria. Targeted SWATH analysis has led to quantification of 13 glycated peptides representing nine lysine sites. These peptides could serve as novel markers in diabetes.     

On approaches to the functional restoration of salivary glands damaged by radiation therapy for head and neck cancer,with a review of related aspects of salivary gland morphology and development

Radiation therapy for cancer of the head and neck can devastate the salivary glands and partially devitalize the mandible and maxilla. As a result, saliva production is drastically reduced and its quality adversely altered. Without diligent home and professional care, the teeth are subject to rapid destruction by caries, necessitating extractions with attendant high risk of necrosis of the supporting bone. Innovative techniques in delivery of radiation therapy and administration of drugs that selectively protect normal tissues can reduce significantly the radiation effects on salivary glands. Nonetheless, many patients still suffer severe oral dryness. I review here the functional morphology and development of salivary glands as these relate to approaches to preventing and restoring radiation-induced loss of salivary function. The acinar cells are responsible for most of the fluid and organic material in saliva, while the larger ducts influence the inorganic content. A central theme of this review is the extent to which the several types of epithelial cells in salivary glands may be pluripotential and the circumstances that may influence their ability to replace cells that have been lost or functionally inactivated due to the effects of radiation. The evidence suggests that the highly differentiated cells of the acini and large ducts of mature glands can replace themselves except when the respective pools of available cells are greatly diminished via apoptosis or necrosis owing to severely stressful events. Under the latter circumstances, relatively undifferentiated cells in the intercalated ducts proliferate and redifferentiate as may be required to replenish the depleted pools. It is likely that some, if not many, acinar cells may de-differentiate into intercalated duct-like cells and thus add to the pool of progenitor cells in such situations. If the stress is heavy doses of radiation, however, the result is not only the death of acinar cells, but also a marked decline in functional differentiation and proliferative capacity of all of the surviving cells, including those with progenitor capability. Restoration of gland function, therefore, seems to require increasing the secretory capacity of the surviving cells, or replacing the acinar cells and their progenitors either in the existing gland remnants or with artificial glands.     

In vitro antimicrobial activity of zimmu ( Allium sativum L. ‘ Allium cepa L.) leaf extract

The fungitoxic effect of various medicinal plants belonging to different families was evaluated in vitro on Rhizoctonia solani, the rice sheath blight pathogen. Of the various plant extracts, the leaf extract of zimmu (Allium cepa × Allium sativum) showed the maximum antifungal activity against R. solani and recorded an inhibition zone of 12?mm. The leaf extract of zimmu was also effective in inhibiting the growth of other agronomically important fungal and bacterial pathogens viz., Aspergillus flavus, Curvularia lunata, Alternaria solani, Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. malvacearum and Xanthomonas axonopodis pv. citri. The antimicrobial compound was dissoluble in methanol and the methanolic extract showed the absorption maxima at 210?nm and 230?nm. Phenolic compounds were present in greater amounts in methanol extract of zimmu. TLC analysis showed the appearance of two blue spots at R _f ?=?0.65 and R _f ?=?0.90. The compounds eluted at R _f ?=?0.65 and R _f ?=?0.90 by preparative TLC exhibited strong antifungal activity against R. solani.     

Ganoderma – a basal stem rot disease of coconut palm in south Asia and Asia pacific regions

Basal stem rot (BSR) caused by the species of Ganoderma is one of the most devastating diseases of numerous perennial, coniferous and palmaceous hosts. In forest systems, Ganoderma has an ecological role in the breakdown or delignification of woody plants. Symptoms of BSR disease can take several years to develop, and the presence of the pathogen (such as indicated by fruiting bodies) is often only visible when the fungus is well established and more than half of the bole tissue has been decayed, leaving no chance for the grower to cure the infected palms. Soils with poor drainage and water stagnation during rainy seasons were found to favour the disease. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. Amendment of calcium nitrate in soil is one of the methods for the management of Ganoderma disease in palms. A combination of biological and systemic fungicides along with good cultural practices will effectively control the BSR disease in palms.     

Anticancer effects and lysosomal acidification in A549 cells by Astaxanthin from Haematococcus lacustris

Astaxanthin (AXN) is known to have health benefits by epidemiological studies. Therefore, it is of interest to assess the effect of AXN (derived from indigenous unicellular green alga Haematococcus lacustris) to modulate cell cycle arrest, lysosomal acidification and eventually apoptosis using in vitro in A549 lung cancer cells. Natural extracts of astaxanthin were obtained by standardized methods as reported earlier and characterized by standard HPLC and MS. Treatment of A549 cells with AXN (purified fraction) showed significant reduction in cell viability (about 50%) as compared to crude extract at 50µM concentration. Thus, we show the anticancer effects and lysosomal acidification in A549 cells by Astaxanthin from Haematococcus lacustris for further consideration. Together, our results demonstrated the anticancer potential of AXN from Haematococcus lacustris, which is found to be mediated via its ability to induce cell cycle arrest, lysosomal acidification and apoptotic induction.     

Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

Anti-ulcer and antioxidant activity of GutGard

The present study was undertaken to determine the anti-ulcer and antioxidant potential of GutGard, a standardized extract of Glycyrrhiza glabra commonly known as licorice. Effect of various doses (12.5, 25, and 50 mg/kg, po) of GutGard was studied on gastric ulcers in pylorus ligation-, cold-restraint stress- and indomethacin induced gastric mucosal injury in rats. Anti-ulcer activity was evaluated by measuring the ulcer index, gastric content, total acidity, and pH of gastric fluid. GutGard dose dependently decreased gastric content, total acidity, ulcer index and increased pH of gastric fluid in pylorus ligation ulcer model. In cold-restraint stress- and indomethacin induced ulcer models all the doses of GutGard decreased the ulcer index and increased the pH of gastric fluid. The antioxidant activity was evaluated by the oxygen radical absorbance capacity (ORAC) assay. GutGardT exhibited potent antioxidant activity with high hydrophilic and lipophilic ORAC value. GutGard possessed anti-ulcerogenic properties that might be afforded via cytoprotective mechanism by virtue of its antioxidant properties. These results supported the ethnomedical uses of licorice in the treatment of gastric ulcer.