A universal expression/silencing vector in plants

A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl virus's natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper "virus," transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.     

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.     

Baculovirus expression and bioactivity of a soluble 140 kDa extracellular cleavage fragment of L1 neural cell adhesion molecule

L1 neural cell adhesion molecule is the founding member of the L1 subfamily of the immunoglobulin superfamily and plays an important role in the overall development of both the central and peripheral nervous systems, making it an attractive candidate for promoting neural regeneration following injury. Currently, L1 used for experimental studies is primarily mammalian-derived; however, the insect cell expression system described here provides an alternative source of recombinant L1 with equivalent bioactivity. A 140 kDa L1 fragment based on a physiological plasmin cleavage site in the extracellular domain was cloned and expressed with a C-terminal 6x histidine tag. Recombinant insect cell-derived L1 was analyzed by Western blot using an antibody to human L1 to confirm immunogenicity and to optimize infection conditions for recombinant L1 production. The recombinant protein was secreted by insect cells, efficiently purified under non-denaturing conditions using dialysis followed by metal affinity chromatography, and analyzed by SDS-PAGE to produce a single band of the expected approximate 140 kDa size. The bioactivity of insect cell-derived L1 was compared to mammalian-derived L1-Fc and poly-L-lysine (PLL) using chick embryonic forebrain neurons. The results show comparable, robust neurite outgrowth at 24h on insect cell-derived L1 and mammalian-derived L1-Fc, with significantly longer neurites than those observed on PLL. Future studies will examine the immobilization of L1 to biomaterial surfaces in physiologically appropriate orientation via the C-terminal 6x histidine tag and will investigate their application in promoting axonal regeneration in the injured nervous system.     

RAGE mRNA expression in the diabetic mouse kidney

Receptors for advanced glycation end products (RAGE), which bind and internalize AGE-modified proteins formed from oxidation and other products of the nonenzymatic glycation reaction, have been mechanistically implicated in the development of the chronic complications of diabetes. In the present experiments, we sought evidence for the participation of RAGE in diabetic nephropathy by analysis of steady state levels of mRNA encoding RAGE in the renal cortex of a well-defined animal model (the db/db mouse) that develops renal pathology similar to that found in human diabetes. In these animals, increased AGE-product formation was confirmed by measurement of fluorescence in serum and renal cortex proteins. Renal involvement was confirmed by demonstration of increased urine albumin excretion and elevated serum creatinine concentrations relative to nondiabetic (db/m) littermate controls. Despite elevated concentrations of circulating and tissue AGE-modified proteins, the level of RAGE mRNA expression in renal cortex of diabetic mice did not significantly differ from that in nondiabetic littermate controls. The findings militate against changes in RAGE expression in the pathogenesis of renal abnormalities in this animal model.     

The nature and identification of quantitative trait loci: a community's view

This white paper by eighty members of the Complex Trait Consortium presents a community's view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?     

Effect of positional changes of anatomic structures on upper airway dilating muscle shortening during electro- and chemostimulation.

Positional changes of anatomic structures surrounding the upper airway are known to affect pharyngeal mechanics and collapsibility. We hypothesized that these alterations also affect the ability of the upper airway dilator muscles to enlarge the pharynx by altering their ability to shorten when activated. Using sonomicrometry, we evaluated in seven anesthetized dogs the effects of changes in tracheal and head position on the length of the genioglossus (GG) and the geniohyoid (GH) and the effects of these positional changes on the magnitude of shortening of the two muscles in response to electro- (ES) and chemostimulation (CS). Caudal traction of the trachea lengthened the GG and GH in all dogs, whereas cranial displacement of the trachea and flexion of the head to a vertical position shortened the muscles. Compared with the magnitude of ES-induced shortening in the neutral position, ES-induced shortening of the GG was 144.7 +/- 14.6, 49.3 +/- 4.3, and 33.5 +/- 11.6% during caudal and cranial displacement of the trachea and during head flexion, respectively. Similar effects of the positional changes were found for the GH, as well as for both muscles during respiratory stimulation with P(CO2) of 90 Torr at the end of CO(2) rebreathing, although inspiratory muscle shortening during CS reached only one-quarter to one-third of the magnitude observed during ES. We conclude that positional alterations of anatomic structures in the neck have a dramatic effect on the magnitude of shortening of the activated GG and GH, which may reduce substantially their ability to protect pharyngeal patency.     

In vitro mitochondrial effects of PK 11195, a synthetic translocator protein 18 kDa (TSPO) ligand,in human osteoblast-like cells

The role of the TSPO in metabolism of human osteoblasts is unknown. We hypothesized that human osteoblast metabolism may be modulated by the TSPO. Therefore we evaluated the presence of TSPO in human osteoblast-like cells and the effect of its synthetic ligand PK 11195 on these cells. The presence of TSPO was determined by [³H]PK 11195 binding using Scatchard analysis: Bmax 7682 fmol/mg, Kd 9.24 nM. PK 11195 did not affect significantly cell proliferation, cell death, cellular viability, maturation, [¹⁸F]-FDG incorporation and hexokinase 2 gene expression or protein levels. PK 11195 exerted a suppressive effect on VDAC1 and caused an increase in TSPO gene expression or protein levels. In parallel there was an increase in mitochondrial mass, mitochondrial ATP content and a reduction in ΔΨm collapse. Thus, it appears that PK11195 (10⁻⁵ M) stimulates mitochondrial activity in human osteoblast-like cells without affecting glycolytic activity and cell death.     

Alpha-adrenoreceptor blockade with phenoxybenzamine does not affect the ability of the nose to condition air.

The primary function of the nose is to warm and humidify air. We have previously shown that raising nasal mucosal temperature by immersing feet in warm water increases the amount of water evaporated by the nose as air passes through it (nasal conditioning capacity; Abbott D, Baroody F, Naureckas E, and Naclerio R. Am J Rhinol 15: 41-45, 2001). To investigate further the effect of nasal mucosal temperature on nasal conditioning capacity, we raised the temperature through alpha-adrenoreceptor blockade by intranasally administering phenoxybenzamine. We hypothesized that blocking alpha-adrenoreceptors during inhalation of cold, dry air would lead to an increase in nasal blood flow, surface temperature, and nasal conditioning capacity, as measured by the water gradient. After appropriate pilot studies, we performed a double-blind, placebo-controlled, two-way crossover study in nine nonatopic, healthy subjects by studying the effect of treatment with intranasal phenoxybenzamine. Nasal mucosal temperature increased significantly after administration of phenoxybenzamine and was associated with a significantly smaller net decrease in nasal mucosal temperature after exposure to cold, dry air (P < 0.05). However, there were no significant differences in nasal conditioning capacity between treatments (P > 0.05). Phenoxybenzamine decreased the symptom of rhinorrhea after exposure to cold, dry air (P < 0.05), but congestion was not different between individuals given phenoxybenzamine and placebo (P > 0.05). Our data demonstrate that phenoxybenzamine, despite raising mucosal temperature and not affecting nasal volume, did not affect the ability of the nose to warm and humidify air.     

Genotype Is a Stronger Determinant than Sex of the Mouse Gut Microbiota

The mammalian gut microbiota is considered to be determined mostly by diet, while the effect of genotype is still controversial. Here, we examined the effect of genotype on the gut microbiota in normal populations, exhibiting only natural polymorphisms, and evaluated this effect in comparison to the effect of sex. DNA fingerprinting approaches were used to profile the gut microbiota of eight different recombinant inbred mouse lines of the collaborative cross consortium, whose level of genetic diversity mimics that of a natural human population. Analyses based on automated ribosomal internal transcribed spacer analysis demonstrated significant higher similarity of the gut microbiota composition within mouse lines than between them or within same-gender groups. Thus, genetic background significantly impacts the microbiota composition and is a stronger determinant than gender. These findings imply that genetic polymorphisms help shape the intestinal microbiota of mammals and consequently could affect host susceptibility to diseases.     

The reductive enzymatic cleavage of thiosulfate

Summary Methods presently used for the enzymatic assay of the thiosulfate cleaving reaction in bacterial cell-free systems are critically examined. Conditions under which strong acids are used to terminate the reaction and to release H₂S proved to be unsuitable. A non-enzymatic production of H₂S under such conditions is demonstrated. A reliable procedure for the measurement of H₂S production from the enzymatic cleavage of thiosulfate is described. This method was used to measure the thiosulfate cleaving reaction catalyzed by cell-free extracts of phototrophic bacteria of the genusChromatium. As reductants, hydrogen-hydrogenase/methyl viologen system, reduced glutathione or dihydrolipoate were used. The same extract fraction catalyzed the rhodanese reaction.