Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.

Effect of Lycopene on Histological Alterations of Basement Membrane & Fibrosis in Renal Interstitial Tissue of Celebrex Induced Albino Rats; An Experimental Study

Objective: To assess the histological alterations in basement membrane and fibrosis in renal interstitium of albino rats due to celebrex with enhancement by lycopene. Study Design: Experimental study. Abode of Study: Animal House of Jinnah Postgraduate institute, Karachi, Materials and Methods: COX-2 inhibitor and antioxidant medicines were used in this research work. These medications were orally administered in 40 male albino rats weighing 200-220gm for experimentation. Rats were housed in separate pens at 23ºC. Rats were arranged into 4 groups including control horde and three experimental hordes. The medications were dose up orally by gastric tube daily for one month. At completion of experiment, animals were dissected and tissues were well-preserved for staining. Results: In second horde PAS stained kidney segments showed disrupted basement membrane of distended proximal convoluted tubules & ill-defined brush border and fibrosis in renal interstitium, but 3rd horde had intact basement membrane & well-define brush border at the luminal surface of proximal tubular epithelium and there was mild fibrosis in renal interstitium. Conclusion: This study divulges that lycopene convalesce the disrupted basement membrane and fibrosis in second horde.

Drosophila renal stem cells enhance fitness by delayed remodeling of adult Malpighian tubules

Drosophila renal stem cells (RSCs) contradict the common expectation that stem cells maintain tissue homeostasis. RSCs are abundant, quiescent and confined to the peri-ureter region of the kidney-like Malpighian tubules (MTs). Although derived during pupation like intestinal stem cells, RSCs initially remodel the larval MTs only near the intestinal junction. However, following adult injury to the ureter by xanthine stones, RSCs remodel the damaged region in a similar manner. Thus, RSCs represent stem cells encoding a developmental redesign. The remodeled tubules have a larger luminal diameter and shorter brush border, changes linked to enhanced stone resistance. However, RSC-mediated modifications also raise salt sensitivity and reduce fecundity. Our results suggest that RSCs arose by arresting developmental progenitors to preserve larval physiology until a time in adulthood when it becomes advantageous to complete development by RSC activation.One-Sentence SummaryActivated Drosophila renal stem cells rebuild the adult Malphigian tubules using a less efficient but more stone-resistant design.

Changes in the Expression of Renal Brush Border Membrane N-Glycome in Model Rats with Chronic Kidney Diseases

Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments.

High resolution dynamic mapping of the C. elegans intestinal brush border

The intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption, and host defense. Studies on mammalian cultured epithelial cells uncovered some of the molecular players and physical constrains required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not been investigated in detail yet. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode C. elegans to build a high-resolution and dynamic localization map of known and new brush border markers. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation but become highly stable once microvilli are built. This new toolbox will be instrumental to understand the molecular processes of microvilli growth and maintenance in vivo as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity.

Critical domains in the specific binding of radiolabelled Vip3Af insecticidal protein to brush border membrane vesicles from Spodoptera spp. and cultured insect cells

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis ). In this study, we have set up the conditions to analyze the specific binding of 125 I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but that Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine-mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125 I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding, and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125 I-DI-III. In addition, the truncated protein 125 I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo. IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for the ex vivo toxicity to Sf21 cells.

Lactose Glycation of the Maillard-Type Impairs the Benefits of Caseinate Digest to the Weaned Rats for Intestinal Morphology and Serum Biochemistry

The Maillard reaction between the lactose and milk proteins unavoidably occurs during the thermal treatment of milk. Although the impact of this reaction on protein nutrition and safety has been well-studied, whether a lactose glycation of milk proteins of the Maillard-type might affect the rats in their growth and intestinal morphology needs an investigation. In this study, caseinate and lactose-glycated caseinate were digested using pepsin and trypsin. Afterward, the resultant caseinate digest and glycated caseinate digest (lactose content of 13.5 g/kg of protein) at 100, 200, and 400 mg/kg body weight (BW)/d were assessed for their effects on the female weaned Wistar rats in terms of daily body weight gain, intestinal morphology, digestive and brush-border enzyme activities, as well as serum chemical indices. The results showed that glycated caseinate digest always showed a weaker effect on rat than caseinate digest either at the 0–7 or 0–28 d feeding stage, and more importantly, at the highest dose of 400 mg/kg BW/d, it caused obvious adverse effect on the rats, reflected by lower values of these indices. Compared with caseinate digest, glycated caseinate digest in the rats caused 0.9–15.4% and 10.6–49.7% decreases in average daily gain of BW and small intestinal length, 1.1–21.5% and 2.3–33.3% decreases in villus height and the ratio of villus height to crypt depth of the small intestine, or 0.3–57.6% and 0.2–55.7% decreases in digestive and critical brush-border enzyme activities, respectively. In addition, when the rats were fed with glycated caseinate digest, some serum indices related to oxidative stress status were enhanced dose-dependently. Lactose glycation of the Maillard-type is thus considered as a negative event of the Maillard reaction on milk proteins because this reaction might impair protein benefits to the body.

Sub-fractions from Carica Papaya Seed Extracts Can Prevent Potassium Bromate- induced Changes in Activities of Renal Brush Border Membrane Enzymes and Some Enzymes of Carbohydrate Metabolism in the Kidney of Rats

Aim: To investigate the effect of the chromatographic fractions of Carica papaya seed on KBrO3 –induced reduction in the activities of renal brush border membrane (BBM) marker enzymes and the changes in activities of some enzymes of carbohydrate metabolism in the kidney of rats. Study Design: twenty male Wistar rats were divided into four groups, five rats per group; normal control, KBrO3 control, papaya fraction control and KBrO3 group administered with 126mg/kg body weight of the most active fraction of partially purified methanol extract of C. papaya for 48 hours. Place and Duration of Study: Department of Biochemistry Laboratory, Faculty of Basic Medical Sciences, Bayero University Kano, Nigeria. Methodology: The activities of renal BBM marker enzymes: γ-glutamyl transferase, alkaline phosphatase, maltase and leucine aminopeptidase were assayed in homogenates of renal cortex and medulla, and in brush border membrane vesicle (BBMV) isolated from cortex using standard methods. Furthermore, activities of the following enzymes representing different pathways of carbohydrate metabolism were determined in renal homogenates: hexokinase (HK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose 6-phosphatase (G6P), fructose 1,6-bisphosphatase (FBP), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME). Results: KBrO3 administration significantly (P<0.05) decreases the activities of all the BBM marker enzymes in renal homogenates and BBMV. It also decreases the activities of MDH, G6P, FBP and G6PD, and significantly increases (P<0.05) that of HK, LDH and ME in renal homogenates however co-administration of most active fraction of C. papaya seed prevented all the KBrO3 -induced changes in these biochemical parameters. Conclusion: Chromatographic fractions of C. papaya seed extract possesses potent phytochemicals that could prevent KBrO3 –induced reduction in activities of renal BBM marker enzymes and the changes in enzymes of carbohydrate metabolism studied and therefore could be analyzed further to isolate the bioactive compounds.