Effects of Echinostoma caproni infections on metallic ions in the intestinal mucosa of ICR mice

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to study metallic ions in the intestinal mucosa of ICR mice infected with Echinostoma caproni and the mucosa of uninfected control mice. Infected mucosa (n = 9 with about 100 mg wet weight per sample) were examined at 2 weeks p.i. in mice that were infected with about 25 worms per host. Uninfected mucosa (n = 9 with about 100 mg wet weight per sample) were examined in the same time frame as the infected mucosa. Five metals were measured in the mucosa by ICP-AES analysis, as follows: calcium, potassium, magnesium, sodium and zinc. There were no significant differences (Student's t-test, P > 0.05) in the concentrations of calcium, potassium or zinc in infected versus uninfected mucosa. The concentration of sodium was significantly greater (P < 0.05) in the mucosa of infected versus uninfected mucosa, but the situation was reversed in regard to magnesium.     

Effects of diet and larval trematode parasitism on lutein and beta-carotene concentrations in planorbid snails as determined by quantitative high performance reversed phase thin layer chromatography

High performance thin layer chromatography (HPTLC) was used to quantify the concentrations of beta-carotene and lutein in Biomphalaria glabrata and Helisoma trivolvis (Colorado and Pennsylvania strains) snails under various conditions. These conditions were: snails fed a lettuce (L) vs. a yolk (Y) diet; B. glabrata infected with Echinostoma caproni vs. uninfected snails; and H. trivolvis (PA) infected with Echinostoma trivolvis vs. uninfected snails. The pigments were extracted from the snail whole bodies and digestive gland-gonad complexes, separated by reversed phase HPTLC, and quantified by densitometric scanning with standard calibration curves. Snails on the L-diet showed significant increases (Student's t-test, P<0.05) in the concentrations of beta-carotene and lutein compared to snails on the Y-diet. Snails infected with echinostomes showed no significant differences (Student's t-test, P>0.05) in the concentrations of lutein and beta-carotene compared to the uninfected cohorts. Our results were compared with previous studies that analyzed beta-carotene and lutein in snails infected with larval trematodes. Variations in the results of our study compared with others reflect intrinsic differences in the larval trematode-snail systems used.     

Free-pool amino acids in Biomphalaria glabrata infected with Echinostoma caproni as determined by thin-layer chromatography

Thin-layer chromatography was used to analyze the free-pool amino acids of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata infected with Echinostoma caproni and uninfected (control) snails. Qualitative analysis revealed the presence of histidine, lysine, serine, alanine, valine, and isoleucine or leucine in all samples. Quantitative analysis of lysine and valine gave mean weight percentages of 0.00699 +/- 0.0022 and 0.00174 +/- 0.00056, respectively, in the DGG of uninfected snails, and 0.00504 +/- 0.0014 and 0.00254 +/- 0.00033, respectively, in the DGG of infected snails. The differences in values between infected and uninfected snails were not statistically significant (Student's t-test, P > 0.05).     

Thin layer chromatographic analysis of glucose and maltose in estivated Biomphalaria glabrata snails and those infected with Schistosoma mansoni

Thin layer chromatography was used to analyze the glucose and maltose concentrations of the digestive gland–gonad complex (DGG) of uninfected-estivated Biomphalaria glabrata snails and estivated B. glabrata patently infected with Schistosoma mansoni. All snails were estivated in a most chamber at a relative humidity of 98 ± 1% and a temperature of 23 ± 1 °C for 14 days. Carbohydrates were extracted from the DGG with 70% aqueous ethanol, and extracts were analyzed on silica gel preadsorbent plates using ethyl acetate–glacial acetic acid–methanol–water (60:15:15:10) mobile phase, α-naphthol–sulfuric acid detection reagent, and quantification by densitometry. The concentrations of glucose and maltose were significantly reduced in both uninfected-estivated snails and infected-estivated snails.     

Nutritional regulation of immunosenescence for heart health

Immunosenescence via increased inflammatory cytokines may play key regulatory roles in facilitating cardiac infections and heart failure. Based upon recent evidence, we hypothesize that cytokine polarization due to aging directly dysregulates fibroblasts, leading to altered cardiac structure and dysfunction. Some dietary fatty acids should ameliorate heightened inflammatory cytokines thereby retarding cardiac pathology, loss of structural collagen and premature death from heart failure. For example, T-helper (Th) 2 cells' cytokine levels are very high in seniors who have increased heart disease due to suppressed resistance to cardiotrophic pathogens. In addition, such inflammatory cytokines deregulate fibroblasts, thus reducing collagen synthesis, weakening muscle structure and heart pump function for heart failure and hypertension. Therefore, supplementation with n-3 polyunsaturated fatty (PUFA) or conjugated linoleic acids, by reducing Th2 and increasing Th1 cytokines, may provide a sensible and widely available means to treat and even prevent excessive inflammatory cytokines and their cardiotoxic effects. On the other hand, dietary n-6 PUFA may promote cytokine polarization in seniors, exacerbating age-related heart dysfunction.     

Effects on the neutral lipid contents of the liver, ileum and serum during experimental schistosomiasis

During infection of vertebrate hosts with Schistosoma mansoni,worm eggs trapped in host tissues induce granulomatous lesions that interfere with normal organ functions. Even though both the liver and the intestine are particularly susceptible to egg-induced tissue damage, little information is available on the pathobiochemical changes induced in these organs during infection. Using a mouse model, we investigated whether the lipid profiles of the liver and ileum were altered significantly as a result of infection. We found that triacylglycerol and cholesteryl ester levels decreased significantly as infection progressed.     

Tissue-Specific and Developmental Effects of the Easily Shocked Mutation on Ethanolamine Kinase Activity and Phospholipid Composition in Drosophila melanogaster

The easily shocked (eas) gene of Drosophila melanogaster encodes ethanolamine kinase (EK), the first step in phosphatidylethanolamine (PE) synthesis via the CDP-ethanolamine pathway Flies mutant for eas display a complex neurological phenotype. In this paper we look at the contribution of EK to lipid metabolism during Drosophila development with the goal of linking the eas biochemical defect with the organismal phenotype. Using a chromatography-based assay, EK activity was detected in wild-type flies throughout development. Most of the activity in the adult was present in heads, which is primarily tissue of neural origin. Flies mutant for eas showed severely reduced levels of activity at each stage assayed. Using standard extraction methods and thin layer chromatography, phospholipid composition was assayed in wholeflies and in heads. While PE levels were decreased significantly in both tissues, heads also had significantly less phosphatidylserine (PS). Therefore, decreases in both phospholipids may play a role in producing the aberrant phenotype in eas flies.     

Elevated Plasma Albumin and Apolipoprotein A-I Oxidation under Suboptimal Specimen Storage Conditions

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC–electrospray ionization–MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at −80 °C, −20 °C, and room temperature and exposed to multiple freeze–thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at −20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze–thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress—including acute myocardial infarction and prostate cancer—demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above −30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.Human serum albumin contains a single free cysteine residue (Cys34) that is susceptible to oxidation via disulfide-bond formation with free cysteine amino acids, resulting in S-cysteinylated (oxidized) albumin (1). Human apolipoprotein A-I (apoA-I)¹ contains three methionine residues (Met86, Met112, and Met148) that can be oxidized to sulfoxides (2–4). The oxidized forms of both of these plasma/serum (P/S) proteins have been proposed as markers of conditions involving oxidative stress (5–9), including atherosclerosis (6–8). These proteins are readily analyzed intact via mass spectrometry in a single run using simple dilute-and-shoot techniques; thus if scientifically suitable, they are well positioned analytically to serve as clinical markers. At least some evidence exists, however, that both albumin (10) and apoA-I (6) are susceptible to artifactual oxidation ex vivo. Notably, the scientific literature in recent years has been relatively quiet with regard to both of these markers. We suspected that spontaneous artifactual oxidation of these proteins ex vivo led to their initial implication as markers of disease, but that the same phenomenon might have confounded efforts to clinically validate them (11, 12). Thus we undertook systematic studies of albumin and apoA-I oxidation ex vivo and found evidence indicating that rather than serving as markers of disease, oxidized albumin and apoA-I may serve as markers for improper handling and storage of blood P/S.Improper biospecimen handling and storage can contribute to sample measurements that do not accurately reflect biological reality in vivo (13–16). This may introduce bias in analytical results, limiting the capacity for meaningful comparisons among patient groups (17–19). Thus careful pre-analytical sample handling is a vital component of both clinical investigation and biomarker research. For clinical assays, parameters that define proper sample handling and storage are generally determined during assay validation and are typically incorporated into laboratory standard operating procedures. In blood P/S-based biomarker development work, however, verification of sample integrity is sometimes overlooked or considered only as an afterthought. Contributing to this phenomenon is that fact that there are no universally accepted, globally applicable endogenous reference markers of P/S integrity. Indeed, there likely does not exist a single, individual marker capable of meeting this broad specification. Nonetheless, identification and standardization of quality control markers that cover this specific scope of application (i.e. proper storage conditions for blood P/S) represent an important goal of biobanking-related research (16, 20).Betsou et al. (16) recently outlined and ranked some of the strongest candidates for use as quality control tools in biomarker research. Within the scope of tools for assessing proper handling and storage of P/S samples, nearly all markers are founded on the quantification of a nominal protein via a molecular-recognition-based assay. As a result, the indication of a loss of specimen integrity lies in an apparent loss of the target protein beyond the normal human reference range. Such loss is often ascribed to “degradation” and in many cases likely happens because of residual proteolytic activity that occurs at temperatures above the sample freezing point. In other cases, loss of the protein marker may be due to misfolding caused by repeated freeze–thaw cycles.Though not frequently discussed, protein “degradation” ex vivo may also have roots in oxidative processes that are capable of disrupting protein–antibody interactions that serve as the basis for protein quantification. It is well known that in the absence of special precautions, disulfide bonds will form spontaneously between cysteine thiols. We have previously shown that this requires only the presence of atmospheric oxygen and trace metals and proceeds through a cysteine sulfenic acid intermediate (21). This mechanism also applies to S-cysteinylation of albumin (22–24), though disulfide exchange with cystine may be operative in P/S as well. Likewise, it is known that methionine-containing proteins and peptides will oxidize to sulfoxides spontaneously in the presence of atmospheric oxygen (25, 26); indeed, artifactual sulfoxidation of methionine residues in peptide-based proteomics work is well known. Oxidative modifications such as these have the potential to disrupt antibody interactions with the oxidized protein, resulting in low readings in molecular-recognition-based assays. Thus protein oxidation merits investigation as a protein “degradation” pathway.As pointed out by Betsou et al. (16), markers that are highly sensitive to variations in specimen storage and handling conditions are likely to be the most useful. A considerable degree of change that occurs rapidly under undesirable conditions to which samples may be exposed, such as the state of being incompletely frozen (which for blood plasma occurs at temperatures above −30 °C (27–30)), is something to be sought after in a biospecimen-integrity marker. Herein we describe simple methods for the simultaneous relative quantification of oxidized albumin and apoA-I and present evidence that albumin and apoA-I can undergo major increases in oxidation ex vivo and thus may be useful markers of P/S specimen integrity.     

Effects of diet on the development of Schistosoma mansoni in Biomphalaria glabrata and on the neutral lipid content of the digestive gland-gonad complex of the snail

In a previous study, when the snail Biomphalaria glabrata was infected with Schistosoma mansoni and maintained on a diet of hen's egg yolk, it produced fully developed cercariae in about one-half the time taken by snails fed Romaine lettuce. Increased lipids were also noted in the snails fed the yolk diet. The purpose of the present study was to further investigate nutritional effects of a high-lipid diet on larval schistosome development and to reexamine the time to cercarial patency in infected snails maintained on either the yolk or lettuce diet and to use high-performance thin-layer chromatography (HPTLC) to analyze the neutral lipids in the digestive gland-gonad complex (DGG) of snails maintained on both diets. Infected snails maintained at 26 C and fed either diet produced fully developed cercariae by 4 wk postinfection (PI). Likewise, infected snails maintained at 23 C and fed either diet produced fully developed cercariae by 6 wk PI. The contention that the yolk diet enhanced the time to cercarial patency was not confirmed. The HPTLC analysis of neutral lipids showed that the DGG of infected snails fed the yolk diet contained significantly greater amounts of free sterols and cholesteryl esters but not triacylglycerols than that of the infected snails fed the lettuce diet.