Gastrointestinal and immunological responses of senior dogs to chicory and mannan-oligosaccharides

Thirty-four senior dogs (pointers 8 - 11 years, beagles 9 - 11 years) were used to evaluate the effects of oligosaccharides on nutritional and immunological characteristics. Dogs were randomly allotted to treatments [1% chicory (CH), 1% mannan-oligosaccharide (MOS), 1% chicory + 1% MOS (CM), or no supplementation (control, CON)] in a parallel design with a 4 week baseline period followed by a 4 week treatment period. Dietary supplementation with MOS or CM tended (P = 0.07) to increase food intake due, in part, to an increase in fermentable fibre and a decrease in energy content of the diet. Although wet faecal output increased (P < 0.05) for dogs supplemented with MOS or CM, when corrected for food intake, no differences were noted. The CM treatment increased (P < 0.05) faecal score (1 = hard and dry, 5 = watery liquid), although these scores remained in a desirable range (3 to 3.5). Chicory supplementation increased (P = 0.07) fat digestibility. Chicory or MOS increased (P  0.05) faecal bifidobacteria concentrations 0.4 and 0.5 log₁₀ cfu/g DM, respectively, compared to the CON, while MOS decreased (P < 0.05) faecal E. coli concentrations. Oligosaccharides did not affect white blood cell (WBC) concentrations, but CH and CM tended to increase (P = 0.10) neutrophil concentrations compared to control dogs. Peripheral lymphocyte concentrations were decreased in dogs supplemented with MOS (P = 0.06) and CM (P < 0.05). Chicory and MOS alter faecal microbial populations and certain indices of the immune system of senior dogs.     

Chemoprotective influence of Zanthoxylum sps. on hepatic carcinogen metabolizing and antioxidant enzymes and skin papillomagenesis in murine model

In the present study, the putative potential of pericarp of dried fruit of Zanthoxylum (Rutaceae Family), a common spice additive in India's west coast cuisines, in protecting against carcinogenesis has been reported. Extract from dried fruit of Zanthoxylum was orally administered to mice at two dose levels: 100 and 200 mg/kg body wt. for 14 days. Results reveal bifunctional nature of Zanthoxylum species as deduced from its potential to induce phase-I and phase-II enzyme activities associated with carcinogen activation and detoxification in the liver of mice. Hepatic glutathione S-transferase and DT-diaphorase were found significantly elevated by the treatment. Zanthoxylum was also effective in augmenting the antioxidant enzyme activities of glutathione peroxidase, superoxide dismutase and catalase albeit significantly by high dose of the extract (P < 0.05; P < 0.01). Reduced glutathione was also significantly elevated in the liver of treated animals (P < 0.05). The present study also investigated peri-initiation application of acetone extract of Zanthoxylum on initiated mouse skin. Results showed a significant reduction in tumor incidence from 68% to 36% (P < 0.05); as well as, a reduction in tumor burden per effective mouse from 3.87 to 0.72 (P < 0.01). Cumulatively, the findings strongly suggest cancer chemopreventive potential of Zanthoxylum sps.     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

Temperature-induced changes in the hydroxy and non-hydroxy fatty acid-containing sphingolipids abundant in the surface membrane of Tetrahymena pyriformis NT-1

Sphingolipids make up 30 to 40 mole % of the phospholipids found in the surface membrane of Tetrahymena pyriformis NT-1. We have identified the two major classes as non-hydroxy fatty acid-containing ceramide-2-aminoethylphosphonate (NCAEP) and alpha-hydroxy fatty acid-containing ceramide-2-aminoethylphosphonate (HCAEP). Both classes were well represented in cells grown at 39 degrees C. At this temperature their principal long chain bases were n-hexadeca-4-sphingenine and n-nonadeca-4-sphingenine. The major fatty acid of NCAEP from 39 degrees C-grown cells was palmitic acid and that of HCAEP was alpha-hydroxypalmitic acid. Cells grown at 15 degrees C contained NCAEP, but only traces of HCAEP. By analyzing the incorporation of [1-14C]palmitic acid into cells growing isothermally or shifted from 15 degrees C to 39 degrees C, we obtained evidence favoring a direct conversion of NCAEP to HCAEP. This conversion was blocked in cells grown at 15 degrees C, causing an accumulation of NCAEP. Tetrahymena is a useful model system for studying the poorly understood alpha-hydroxylation process that is of critical importance in myelination of animal nervous tissues.     

Atrazine movement in soil: Comparison of field observations and PRZM simulations

The predictive capability of the pesticide root zone model (PRZM) was investigated for herbicide atrazine [2‐chloro‐4‐(ethylamino)‐6‐(isopropylamino)‐s‐triazine] in corn production under no‐till (NT) and conventional‐till (CT) management practices. Simulation values of atrazine residues obtained using our site‐specific soil and environmental data were compared with the actual values measured in soil samples taken from the root zones of the NT and CT plots during three growing seasons: 1986, 1987, and 1988. The mean concentration of atrazine in soil at each sampling time and depth after application, for each tillage treatment plot (NT or CT), was estimated based on the type of distribution (i.e., normal or lognormal). Overall, the PRZMs simulated concentrations for the top 10 cm of soil compared well with the atrazine residues measured in the CT plots, but overestimated measurements in NT plots. For example, in 1986 the mean atrazine concentration measured in soil samples taken 6 d after application from the top 10 cm of CT plots was 548 μg/kg (S.E. 198 μg/kg), and the PRZM predicted value was 690 μg/kg. In contrast, the mean atrazine concentration for the same soil depth increment in NT plots was 385 μg/kg (S.E. 154 μg/kg), with a PRZM predicted value of 674 μg/kg. Although the PRZM prediction was closer to the measured mean for atrazine concentrations in the top 10 cm of the CT system, the model did not transport atrazine to the lower soil depths, as the actual values have indicated in all 3 years. The results of this model comparison, especially for the lower soil depths (20 to 30 cm) in the NT practice, indicated that the PRZM model does not account for the preferential transport of, and, consequently, underestimates the atrazine residue levels in the lower soil profile under NT management systems.     

Prediction of heterosis in rice based on divergence of morphological and molecular markers

Identifying the best performing hybrid without a field test was essential to save resources and time. In this study, the genetic divergence was estimated using morphological and expressed sequence tag (EST)-derived simple sequence repeats (SSR) markers. Cluster analysis showed that APMS6A and RPHR 1005 belong to groups I and II, respectively, and the hybrid combination recorded the highest mean grain yield of 32.25 g among generated 40 \(\hbox {F}_{1}\hbox {s}\) with standard heterosis of 8.4% over hybrid check, KRH2. The coefficient of marker polymorphism (CMP) value was calculated based on EST-SSR markers; it ranged from 0.40 to 0.80, and a higher CMP value of 0.80 was obtained for the parental combination APMS6A \(\times \) RPHR1005. We predicted heterosis for 40 \(\hbox {F}_{1}\hbox {s}\) based on correlation between CMP and standard heterosis in different traits with standard varietal and hybrid checks indicating positive correlation and significant value for grain yield per plant (\(r=0.58\)**), productivity per day (\(r=0.54\)**), productive tillers (\(r=0.34\)*) and panicle weight (\(r=0.42\)**). This study revealed that the relationship of molecular marker heterozygosity, along with the combining ability, high mean value of different traits, grouping of parental lines based on morphological and molecular characterization is helpful to identify heterotic patterns in rice.     

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.     

Quantifying conformational changes in GPCRs: glimpse of a common functional mechanism

Background

G-protein-coupled receptors (GPCRs) are important drug targets and a better understanding of their molecular mechanisms would be desirable. The crystallization rate of GPCRs has accelerated in recent years as techniques have become more sophisticated, particularly with respect to Class A GPCRs interacting with G-proteins. These developments have made it possible for a quantitative analysis of GPCR geometrical features and binding-site conformations, including a statistical comparison between Class A GPCRs in active (agonist-bound) and inactive (antagonist-bound) states.

Results

Here we implement algorithms for the analysis of interhelical angles, distances, interactions and binding-site volumes in the transmembrane domains of 25 Class A GPCRs (7 active and 18 inactive). Two interhelical angles change in a statistically significant way between average inactive and active states: TM3-TM6 (by -9°) and TM6-TM7 (by +12°). A third interhelical angle: TM5-TM6 shows a trend, changing by -9°. In the transition from inactive to active states, average van der Waals interactions between TM3 and TM7 significantly increase as the average distance between them decreases by >2 Å. Average H-bonding between TM3 and TM6 decreases but is seemingly compensated by an increase in H-bonding between TM5 and TM6. In five Class A GPCRs, crystallized in both active and inactive states, increased H-bonding of agonists to TM6 and TM7, relative to antagonists, is observed. These protein-agonist interactions likely favour a change in the TM6-TM7 angle, which creates a narrowing in the binding pocket of activated receptors and an average ~200 ų reduction in volume.

Conclusions

In terms of similar conformational changes and agonist binding pattern, Class A GPCRs appear to share a common mechanism of activation, which can be exploited in future drug development.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0567-3) contains supplementary material, which is available to authorized users.     

Isomerization of non-insecticidal α-hexachlorocyclohexane (HCH) to insecticidal γ-HCH by Pseudomonas strain Ptm+

Pseudomonas strain Ptm⁺ grew on α-hexa-chlorocyclohexane (HCH, CAS no. 319846), using it as the sole source of carbon and energy. In a replacement-culture study, with the non-insecticidal α-HCH, γ-HCH (CAS no. 58899) was the first metabolite noticed at 6 h, and transient accumulation of insecticidal γ-HCH occurred for up to 18 h. Although delta- (CAS no. 319868) and beta-isomers (CAS no. 319857) were also detected, their concentrations were very low. By 18 h of incubation, about 23% of the α-HCH added was transformed into the gamma-isomer. Subsequently, the concentration of γ-HCH in the medium fell. Thin-layer chromatography, gas chromatography, gas chromatography-mass spectrometry and mosquito-larval bioassay analyses confirmed the formation of γ-HCH. This was associated with the formation of three more metabolites. Received: 6 April 1999 / Received revision: 3 August 1999 / Accepted: 6 August 1999