Physiological Changes in Cultured Sorghum Cells in Response to Induced Water Stress : II. Soluble Carbohydrates and Organic Acids

Eight cultivars Sorghum bicolor (L.) Moench were grown as callus cultures under induced, prolonged water stress (8 weeks), with polyethylene glycol in the medium. Concentrations of soluble carbohydrates and organic acids in callus were measured at the end of the growth period to determine differences in response to prolonged water stress. Sucrose, glucose, fructose, and malate were the predominant solutes detected in all callus at all water potentials. All cultivars had high levels of solutes in the absence of water stress and low levels in the presence of prolonged water stress. However, at low water potentials, low levels of solutes were observed in drought-tolerant cultivar callus and high solute levels were observed in drought-susceptible cultivar callus. Estimated sucrose concentrations were significantly higher in water-stressed, susceptible cultivar callus. Large solute concentrations in susceptible cultivar callus were attributed to osmotic adjustment and/or reduced growth during water stress.     

A metabolic signature for long life in the Caenorhabditis elegans Mit mutants

Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial‐derived α‐ketoacids and α‐hydroxyacids that are produced by long‐lived Mit mutants but not by other long‐lived mutants or by short‐lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α‐ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild‐type animals was reduced using RNA interference we observed an unprecedented effect on lifespan – as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype.     

A role for copper in protozoan grazing – two billion years selecting for bacterial copper resistance

The Great Oxidation Event resulted in integration of soft metals in a wide range of biochemical processes including, in our opinion, killing of bacteria by protozoa. Compared to pressure from anthropologic copper contamination, little is known on impacts of protozoan predation on maintenance of copper resistance determinants in bacteria. To evaluate the role of copper and other soft metals in predatory mechanisms of protozoa, we examined survival of bacteria mutated in different transition metal efflux or uptake systems in the social amoeba Dictyostelium discoideum. Our data demonstrated a strong correlation between the presence of copper/zinc efflux as well as iron/manganese uptake, and bacterial survival in amoebae. The growth of protozoa, in turn, was dependent on bacterial copper sensitivity. The phagocytosis of bacteria induced upregulation of Dictyostelium genes encoding the copper uptake transporter p80 and a triad of Cu(I)‐translocating P_IB‐type ATPases. Accumulated Cu(I) in Dictyostelium was monitored using a copper biosensor bacterial strain. Altogether, our data demonstrate that Cu(I) is ultimately involved in protozoan predation of bacteria, supporting our hypothesis that protozoan grazing selected for the presence of copper resistance determinants for about two billion years.     

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.     

Investigations on the venom of the South Indian scorpion Heterometrus scaber

The enzymes from the venom of Heterometrus scaber, the indole compounds present and the toxic protein of the venom have been studied. The venom contains acid phosphatase, ribonuclease, 5′-nucleotidase, hyaluronidase, acetylcholine esterase and phospholipase A. The indole compounds present in the venom have been identified as 5-hydroxytryptophan, tryptophan, serotonin and tryptamine, along with two unidentified indole compounds. The venom produces hyperglycaemia in sublethal doses and this has been found to be due to increased adrenaline secretion. The toxic protein of the venom has been obtained in a pure form by (NH₄)₂ SO₄ fractionation, followed by fractional precipitation with acetone and chromatography over DEAE-Sephadex. The toxic fraction has been found to be homogeneous on acrylamide gel electrophoresis. It is a glycoprotein (molecular weight 15 000) containing 1.74% glucosamine, 0.87% galactosamine, 0.313% sialic acid, 3.25% fucose and 0.45% of an unidentified neutral sugar. It did not show any enzyme activities, haemolytic activity or inhibition of succinate dehydrogenase activity but it produced hyperglycaemia in sublethal doses. The toxic level (intravenous administration in rats) was found to be 0.72 mg/kg body weight.     

Plasma Osmolality Predicts Extracellular Fluid Catechol Concentrations in the Lateral Hypothalamus

The lateral hypothalamus has an important role in regulating food and water intake. We have investigated the endogenous release of monoamines from the lateral hypothalamus during manipulations of plasma osmolality and circulating volume. Adult male Sprague-Dawley rats implanted with carbon paste in vivo electrochemical (EC) electrodes in the lateral hypothalamus were placed on a 72-h water deprivation schedule. Although the carbon paste EC electrode has an intrinsically ambiguous signal in which changes in ascorbic acid may appear as changes in catechol concentrations, pharmacologic studies in lateral hypothalamus indicated that the electrode most likely measured norepinephrine and possibly epinephrine. On the test day, the EC electrodes were scanned with linear sweep voltammetry from -0.2 to +0.4 V at a rate of 5 mV/s. Semiderivative signal processing showed catechol and hydroxyindole peaks at +0.11 and +0.23 V, respectively. Baseline recordings were made prior to rats drinking distilled water, 10% sucrose, 5% dextrose, 0.30% NaCl, 0.90% NaCl, or 10% d-mannitol. To control for the act of drinking, other implanted dehydrated rats were intraperitoneally injected with 5% dextrose, 0.30% NaCl, or 0.90% NaCl. To dissociate the effects of osmolality and circulating volume on the EC response, hydrated rats implanted with EC electrodes were subcutaneously injected with 12% NaCl or intraperitoneally injected with 35% polyethylene glycol. Other rats subjected to water deprivation and osmotic challenges were decapitated and trunk blood was collected for measurements of plasma osmolality and hematocrit. Similar experiments were conducted using homozygous Brattleboro rats which lack arginine vasopressin (AVP) but which preserve normal plasma osmolality with prodigious drinking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Promiscuous binding at the crossroads of numerous cancer pathways: insight from the binding of glutaminase interacting protein with glutaminase L

The glutaminase interacting protein (GIP) is composed of a single PDZ domain that interacts with a growing list of partner proteins, including glutaminase L, that are involved in a number of cell signaling and cancer pathways. Therefore, GIP makes a good target for structure-based drug design. Here, we report the solution structures of both free GIP and GIP bound to the C-terminal peptide analogue of glutaminase L. This is the first reported nuclear magnetic resonance structure of GIP in a complex with one of its binding partners. Our analysis of both free GIP and GIP in a complex with the glutaminase L peptide provides important insights into how a promiscuous binding domain can have affinity for multiple binding partners. Through a detailed chemical shift perturbation analysis and backbone dynamics studies, we demonstrate here that the binding of the glutaminase L peptide to GIP is an allosteric event. Taken together, the insights reported here lay the groundwork for the future development of a specific inhibitor for GIP.