Potentiation of the natriuretic effect of clonidine following indomethacin in the rat.

Previous studies have demonstrated a diuretic effect of clonidine at low intrarenal infusion rates with a natriuretic effect being observed at high infusion rates (greater than or equal to 3 micrograms.kg-1.min-1). The natriuresis at high infusion rates may have been secondary to increased renal prostaglandin production. We therefore evaluated the effects of indomethacin (a cyclooxygenase inhibitor) on the response to clonidine in the anesthetized rat. Intrarenal infusions of saline (vehicle) or clonidine (0.1, 0.3, 1, and 3 micrograms.kg-1.min-1) were examined both in the presence and absence of pretreatment with indomethacin (5 mg/kg, i.p.). Clonidine produced a dose-related increase in urine volume and free water clearance at 0.3, 1, and 3 micrograms.kg-1.min-1 as compared with the vehicle group. Sodium excretion and osmolar excretion were increased only at the highest infusion rate investigated. Following indomethacin pretreatment, clonidine produced a greater increase in urine volume at each infusion rate investigated. The indomethacin pretreatment also resulted in a potentiation of the natriuretic effect of clonidine at all infusion rates. Interestingly, this was associated with an increase in osmolar clearance but not free water clearance. These effects of indomethacin were reversed by infusion of prostaglandin E2. An infusion of prostaglandin E2 attenuated the indomethacin-induced increase in both urine flow rate and sodium excretion, indicating that the effects of indomethacin were mediated by prostaglandin inhibition. These results suggest that endogenous prostaglandin production attenuates the renal effects of clonidine, and as well, that in the presence of alpha 2-adrenoceptor stimulation, prostaglandin E2 mediates an antidiuretic and antinatriuretic effect.     

Array-based gene discovery with three unrelated subjects shows SCARB2/LIMP-2 deficiency causes myoclonus epilepsy and glomerulosclerosis

Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies.     

Factors affecting overwinter survival of the American burying beetle, <Emphasis Type="Italic">Nicrophorus americanus</Emphasis> (Coleoptera: Silphidae)

The endangered American burying beetle (Nicrophorus americanus) is relatively abundant at Fort Chaffee Maneuver Training Center in northwestern Arkansas. There is a paucity of basic life-history information available, particularly with respect to factors affecting overwintering success. In a field experiment we: (1) captured beetles at Fort Chaffee; (2) bred them in captivity; (3) in the fall on Fort Chaffee placed offspring individually in well-ventilated, lidded 21.1-l buckets containing original soil plugs in grassland or woodland, either provisioned or not with a rat carcass as potential food; (4) overwintered the beetles; (5) checked in the spring to determine survival; and (6) released surviving beetles. Overall, 59.6% of 104 beetles survived the winter, with 77.1% and 44.6% survival in provisioned and nonprovisioned buckets, respectively. No differences were evident between habitats. Beetle age was an important survival predictor, with older beetles having a higher survival probability, but only if nonprovisioned. Gender and body size were not predictive of survival. Many surviving beetles were at or near the surface; depth averaged 6.0 cm, with some as deep as 20 cm. Our findings suggest that American burying beetles will have a higher probability of overwinter survival if carcasses are readily available as winter approaches.     

3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin.

The type II chaperonin CCT (chaperonin containing Tcp-1) of eukaryotic cytosol is a heteromeric 16-mer particle composed of eight different subunits. Three-dimensional reconstructions of apo-CCT and ATP-CCT have been obtained at 28 A resolution by cryo-electron microscopy. Binding of ATP generates an asymmetric particle; one ring has a slightly different conformation from the apo-CCT ring, while the other has undergone substantial movements in the apical domains. Upon ATP binding the apical domains rotate and point towards the cylinder axis, so that the helical protrusions present at their tips could act as a lid closing the ring cavity.     

Labeling to manage marketing of GM foods

Biotechnology has the potential to introduce new food safety risks, liabilities and benefits, and although privately managed supply chains (involving proactive management of the production of branded products) are effective at providing, managing and communicating adequate information about products with well understood risks, products with uncertain risks pose a greater challenge. The demand for increased product information regarding genetically modified content, in particular, places new constraints on food supply chains, frequently resulting in communication failures. Here we assess and reject mandatory labeling as an appropriate response.     

Effect of reproductive status on uptake of latex microparticles in rat small intestine

This study investigates whether pregnancy or lactation affects microparticle uptake across the small intestinal mucosal barrier, since aspects of gastrointestinal physiology such as motility may be altered in these conditions. It also reports on validation of the model by several methods and discusses the findings in relation to possible mechanisms. Anaesthetised, pregnant, lactating, virgin female or male adult rats were gavaged with fluorescent latex microparticles. The small intestine was removed and fixed either 5 or 30 min later and successive segments of equal length were examined with fluorescence microscopy. Minor adjustments were made to experimental methods to explore details of the uptake mechanism. Control sections contained no particles. All experimental samples showed luminal and surface particles and also contained particles within the tissue, most associated with villous absorptive enterocytes. Particle uptake was greatest at the 30-min time-point, when maximum uptake was usually in the proximal jejunum; although in the early lactating group, this was shifted distally. Total tissue uptake was increased in pregnant and early lactating groups, mainly at villous absorptive and mucus-secreting cells. Accumulation and progression of particles was reflected in increased numbers in the lamina propria. These data were validated by several methods, including particle detection in the blood and mesenteric lymph nodes in some groups. At both time-points, uptake profiles for pregnancy and early lactation differed from those of other groups, implying possible links between particle uptake and hormone levels, surface mucus and tight junction patency.     

Calcium-dependent plasma membrane binding and cell lysis by perforin are mediated through its C2 domain: A critical role for aspartate residues 429, 435, 483, and 485 but not 491

The lymphocyte pore-forming protein perforin is essential for maintaining immune homeostasis and for effective defense against intracellular pathogens. To date, there have been no reported structure-function studies to substantiate the function of any putative domains of perforin, which have been postulated totally on primary sequence similarities with domains in other proteins. In this report, we have used recently developed modalities for expressing full-length perforin and robust functional assays to investigate one of the hallmarks of perforin function: its absolute dependence on calcium for lipid binding and cell lysis. We provide, for the first time, experimental evidence that the predicted C-terminal C2 motif constitutes a functional domain that is responsible for membrane binding of perforin. Whereas conserved aspartate residues at positions 429, 435, 483, and 485 were essential for calcium-dependent plasma membrane binding and cell lysis, the contribution of Asp-491 was limited. Finally, after experimentally verifying an optimized three-dimensional model, we have made predictions on the impact of two inherited perforin mutations of the C2 domain on calcium-dependent lipid binding and cell lysis.