Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops

A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.     

Cholesterol, cardiolipin, and mitochondria permeabilisation

Apoptosis is a form of programmed cell death required for the development and for the proper functioning of multicellular organisms. It is defined by a combination of morphological and biochemical modifications that result from the activation of a family of proteases called caspases. Several pathways can lead to caspase activation and they often involve the release of apoptogenic factors normally sequestered in the mitochondrial intermembrane space. Complete release of mitochondrial pro-apoptotic factors ultimately results in cell death, whether in a caspase-dependent or independent manner. A tight control of mitochondrial permeability is therefore essential. Mutations of regulators of the process, such as proteins of the Bcl-2 family, have indeed been reported in many cancers. In addition, the contributions of lipids, both as regulators of protein activities and as components of the pore itself, are starting to be unravelled. Early on, the role of the mitochondria-specific phospholipid cardiolipin as a targeting signal for pro-apoptotic proteins of the Bcl-2 family was discovered. This role was then expanded since it was shown that cardiolipin also supports conformational changes undergone by proteins of the Bcl-2 family, serves as a docking station for additional pro-apoptotic factors, and is essential for the permeabilisation of synthetic liposomes by activated Bax and Bak. More recently, cholesterol, whose level is increased in most cancer cells, was shown to contribute to their resistance to cytotoxic stresses. Reducing cholesterol levels might therefore represent an interesting novel target to sensitize cancer cells to chemotherapeutic agents.     

Reactive rise in blood pressure upon cuff inflation: cuff inflation at the arm causes a greater rise in pressure than at the wrist in hypertensive patients

OBJECTIVE: Cuff inflation at the arm is known to cause an instantaneous rise in blood pressure, which might be due to the discomfort of the procedure and might interfere with the precision of the blood pressure measurement. In this study, we compared the reactive rise in blood pressure induced by cuff inflation when the cuff was placed at the upper arm level and at the wrist. PARTICIPANTS AND METHODS: The reactive rise in systolic and diastolic blood pressure to cuff inflation was measured in 34 normotensive participants and 34 hypertensive patients. Each participant was equipped with two cuffs, one around the right upper arm (OMRON HEM-CR19, 22-32 cm) and one around the right wrist (OMRON HEM-CS 19, 17-22 cm; Omron Health Care Europe BV, Hoofddorp, The Netherlands). The cuffs were inflated in a double random order (maximal cuff pressure and position of the cuff) with two maximal cuff pressures: 180 and 240 mmHg. The cuffs were linked to an oscillometric device (OMRON HEM 907; Omron Health Care). Simultaneously, blood pressure was measured continuously at the middle finger of the left hand using photoplethysmography. Three measurements were made at each level of blood pressure at the arm and at the wrist, and the sequence of measurements was randomized. RESULTS: In normotensive participants, no significant difference was observed in the reactive rise in blood pressure when the cuff was inflated either at the arm or at the wrist irrespective of the level of cuff inflation.Inflating a cuff at the arm, however, induced a significantly greater rise in blood pressure than inflating it at the wrist in hypertensive participants for both systolic and diastolic pressures (P<0.01), and at both levels of cuff inflation. The blood pressure response to cuff inflation was independent of baseline blood pressure. CONCLUSIONS: The results show that in hypertensive patients, cuff inflation at the wrist produces a smaller reactive rise in blood pressure. The difference between the arm and the wrist is independent of the patient's level of blood pressure.     

Reduced hippocampal 5HT1A PET receptor binding and depression in temporal lobe epilepsy

Summary:  Purpose: To study the relation of hippocampal 5HT1A receptor binding to symptoms of depression in patients with temporal lobe epilepsy. Depression is common in people with epilepsy, and reduced 5HT1A binding has been reported in patients with primary depressive disorders.
Methods: We studied 45 patients with temporal lobe epilepsy confirmed by ictal video-EEG recording. Mood was assessed with the Beck Depression Inventory (BDI). Positron emission tomographic measurement of 5HT1A receptors was performed with 18F-FCWAY, a highly specific silent antagonist. 3D-T1-weighted MRI was used to correct for structural atrophy. Receptor distribution volume (V) was corrected for plasma tracer free fraction (f1).
Results: There was a significant inverse relation between ipsilateral hippocampal v/f1 and the BDI. For contralateral hippocampus, there was a nonsignificant trend. Patients with BDI > 20 had significantly lower ipsilateral hippocampal V/f1 than patients in the low and medium groups. There was no significant effect of the presence of mesial temporal sclerosis, focus laterality, or gender on the BDI.
Conclusions: Our study shows a relationship between hippocampal 5HT1A binding and depressive symptoms measured by the BDI in patients with epilepsy. The findings parallel results in patients with MDD.     

Decreased cognitive functioning in patients with advanced oral squamous cell carcinoma

Cognitive impairment causes a delay in diagnosis and treatment of the various cancer entities, resulting in reduced surgical outcomes and patient survival. However, no investigations have been carried out as to whether an association exists between cognitive functioning and tumour size in patients with oral squamous cell carcinoma (OSCC). In this study, 46 patients with OSCC were evaluated by using a screening test for dementia, consisting of a combination of the mini-mental state examination and the clock test (81% sensitivity and 90% specificity). Test scores were correlated with tumour size according to the TNM staging system, which was categorized as being either limited (T1, T2; n=24) or advanced (T3, T4; n=22). No difference in age (P=0.172), sex (P=0.330), the percentage of drinkers (P=0.090) or the percentage of smokers (P=0.484) was evident between the groups. Patients with advanced tumour size scored significantly lower (median 5.5 of 9 possible points) when compared with those having tumours of a limited size (median 9 of 9 possible points; P=0.005). The median score of patients with T3/T4 tumours suggested the need for comprehensive neuropsychological evaluations for dementia. In conclusion, this study has demonstrated the correlation of reduced cognitive functioning in patients with advanced OSCC. As a consequence, instructions for the identification of early signs and of symptoms of oral cancer are strongly recommended for relatives and nursing staff of patients with cognitive impairment. Such patients might need immediate treatment for oral cancer but might not be able to understand the significance of their symptoms and therefore present late, often too late.     

Calcium-sensing receptor attenuates AVP-induced aquaporin-2 expression via a calmodulin-dependent mechanism

Recent evidence suggests that arginine vasopressin (AVP)-dependent aquaporin-2 expression is modulated by the extracellular calcium-sensing receptor (CaSR) in principal cells of the collecting duct, but the signaling pathways mediating this effect are unknown. Using a mouse cortical collecting duct cell line (mpkCCD(cl4)), we found that increasing the concentration of apical extracellular calcium or treating with the CaSR agonists neomycin or Gd(3+) attenuated AVP-dependent accumulation of aquaporin-2 mRNA and protein; CaSR gene-silencing prevented this effect. Calcium reduced the AVP-induced accumulation of cAMP, but this did not occur by increased degradation of cAMP by phosphodiesterases or by direct inhibition of adenylate cyclase. Notably, the effect of extracellular calcium on AVP-dependent aquaporin-2 expression was prevented by inhibition of calmodulin. In summary, our results show that high concentrations of extracellular calcium attenuate AVP-induced aquaporin-2 expression by activating the CaSR and reducing coupling efficiency between V(2) receptor and adenylate cyclase via a calmodulin-dependent mechanism in cultured cortical collecting duct cells.     

Bypassing vasopressin receptor signaling pathways in nephrogenic diabetes insipidus

Water reabsorption in the kidney represents a critical physiological event in the maintenance of body water homeostasis. This highly regulated process relies largely on vasopressin (VP) action and on the VP-sensitive water channel (AQP2) that is expressed in principal cells of the kidney collecting duct. Defects in the VP signaling pathway and/or in AQP2 cell surface expression can lead to an inappropriate reduction in renal water reabsorption and the development of nephrogenic diabetes insipidus, a disease characterized by polyuria and polydipsia. This review focuses on the major regulatory steps that are involved in AQP2 trafficking and function. Specifically, we begin with a discussion on VP-receptor-independent mechanisms of AQP2 trafficking, with special emphasis on the nitric oxide-cyclic guanosine monophosphate signaling pathway, followed by a review of the mechanisms that govern AQP2 endocytosis and exocytosis. We then discuss emerging data illustrating roles played by the actin cytoskeleton on AQP2 trafficking, and lastly we consider elements that affect AQP2 protein expression in cells. Recent advances in each topic are summarized and are presented in the context of their potential to serve as a basis for the development of novel therapies that may ultimately improve life quality of nephrogenic diabetes insipidus patients.     

Effects of the renal medullary pH and ionic environment on vasopressin binding and signaling

The kidney has a cortico-medullary interstitial gradient of decreasing pH and increasing concentrations of sodium chloride and urea, but the influence of these gradients on receptor signaling is largely unknown. Here, we measured G-protein coupled receptor function in LLC-PK1 cells acutely exposed to conditions mimicking different kidney regions. Signaling through the parathyroid hormone receptor, normally expressed in the cortex, was greatly reduced at an acidic pH similar to that of the inner medulla. Parathyroid hormone receptor, tagged with green fluorescent protein, showed no ligand-induced internalization. In contrast, under both acidic and hyperosmotic conditions, vasopressin increased intracellular cAMP, and upon binding to its type 2 receptor (V2R) was internalized and degraded. Dose-displacement binding assays with selective vasopressin/oxytocin receptor ligands under inner medullary conditions indicated a shift in the V2R pharmacological profile. Oxytocin did not bind to the V2R, as it does under normal conditions and the vasopressin type 1 receptor (V1R) had reduced affinity for vasopressin compared to the V2R in low pH and high osmolality. We suggest that the cortico-medullary gradient causes a receptor-specific selectivity in ligand binding that is of functional significance to the kidney. While the gradient is important for urinary concentration, it may also play a substantial role in fine-tuning of the vasopressin response through the V2R.