An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p

Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps. Here we present the structure of the NH2-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140). The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin. Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain. The NH2-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes.     

In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene

Transgenic mice carrying the gamma 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of beta-galactosidase activity. These results suggest that gamma 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse gamma 2-crystallin gene. In a broader context, this study also demonstrates the utility of beta-galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.     

Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.     

Arsenic accumulation by rice grown in soil treated with roxarsone

Poultry litter is widely used as a fertilizer for lowland rice in Taiwan and China. However, the organic‐arsenic compound roxarsone (additive of poultry feed) in poultry litter can be absorbed by the plants and the resulting arsenic (As) contamination may pose a serious threat to human health. This study used various amounts of poultry litter contaminated with roxarsone in pot experiments to evaluate the effect of roxarsone on rice agronomic parameters and the bioaccumulation of total and inorganic As in rice‐plant tissues. Rice‐grain yield decreased significantly with increasing As content of the soil, and the critical threshold that killed rice was 200 mg roxarsone (kg soil)^–1. The As concentrations in root, straw, leaf, husk, and grain increased with increasing soil As (p < 1%). At 100 mg roxarsone per kg of soil, the As concentration in the rice grain exceeded the statutory permissible limit of 1.0 mg As (kg dry weight)^–1 and at 25 mg roxarsone (kg soil)^–1, the inorganic As concentrations in grains exceeded the statutory limit of 0.15 mg of inorganic As kg^–1 in China. For all treatments, the As concentrations in various plant tissues at maturity follow the order: root > stem > leaf > husk > grain. Arsenite was the predominant species in root, straw, and grain, while arsenate was the predominant species in leaf and husk. No significant difference existed between the amounts of arsenite and arsenate when various amounts of poultry litter were applied. This result illustrates that large amounts of added roxarsone are not only toxic to rice but also accumulate in grains in the inorganic As forms, potentially posing a threat to human health via the food chain.     

Longitudinal hardness and Young's modulus of spruce tracheid secondary walls using nanoindentation technique

Summary Using a mechanical properties microprobe, measurements of hardness and elastic modulus of tracheid walls in the longitudinal direction of spruce wood were obtained by continuously measuring force and displacement as a diamond indenter impressed a cell wall. Maximum mechanical properties were found at the edges of the walls of angular shaped tracheids. Both the hardness and elastic modulus of latewood cell walls were higher than cell walls in the earlywood. The high spatial resolution of this new concept of mechanical testing allows a direct comparison with ultrastructural and microchemical parameters of lignified cells which opens a wider area of applications for the understanding of intrinsic wood properties.This work was conducted while the senior author was a Visiting Scientist at the Oak Ridge National Lab, Oak Ridge, TN 37831, USA partly with joint fundings from the Austrian Science Foundation (Schrödinger scholarship J799-BIO)     

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification of DNA by PCR has revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real-time PCR detection assays targeting DNA of all four Phytophthora ramorum lineages, and a closely related species, P. lateralis. Nine assays were generated: three targeting DNA of all P. ramorum lineages, one for each lineage of P. ramorum, one for P. lateralis and one targeting DNA of P. ramorum and P. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection of DNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non-target species.     

Identification of the cystic fibrosis gene: genetic analysis

Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level has important implications for genetic diagnosis.     

Digestion Methods for Total Heavy Metals in Sediments and Soils

The objectives of this study were to analyze the total contents of Cd, Cr, Cu, Ni, Pb, and Zn in the freshwater sediments and the arable and non-arable soils of Taiwan, and to compare the different digestion methods for their determination. Two hundred and thirty-nine freshwater sediments were collected from the Fei-Tsui Reservoir Watershed (FTRW) in northern Taiwan. Forty-two surface (0–15 cm) and subsurface (15–30 cm) soil samples were likewise collected from 21 representative arable soils derived from various parent materials and with varying weathered degrees in different regions of Taiwan. In addition, one hundred and ninety-four non-arable soils were collected from the different forest regions, industrial parks, resident areas, and commercial areas. Several digestion methods including the aquaregia and different combinations of concentrated acids (HClO₄, HNO₃, H₂SO₄, or HF) were compared. All samples were digested both by the aqua regia method and the Baker and Amacher method for Cd, Cr, Cu, Ni, Pb, and Zn. The Reisenauer method was used for Cr and the Burau method for Pb further. The results indicate that the best digestion methods to analyze the total contents of heavy metals in the sediments and soils were recommended as follows: the Baker and Amacher method for Cd, Cr,Cu, Ni, and Zn; the Reisenauer method for Cr, but for simplicity the Baker and Amacher method is also recommended as the flexible method for the total analysis of Cr; the aqua regia method for Cu, Ni, and Zn; and the Burau method for Pb.     

Rapid determination of total cholesterol in homogenized milk

A rapid method that is amenable to automation has been developed for the determination of total cholesterol in homogenized milk. The milk sample is saponified in ethanolic KOH in the presence of an internal standard, cholestane. Cholesterol and the internal standard are then isolated by solid-phase extraction on a nonpolar adsorbent and eluted with organic solvent. The evaporated extract is derivatized and analyzed by capillary gas chromatography. Average recovery of cholesterol acetate added to milk prior to saponification was 95%. The average relative standard deviation for repeated analyses was 2%. The limit of detection for this method is 2 mg/100 g. Twenty samples can be analyzed by one analyst in a normal work day if the gas chromatograph is equipped with an autosampler. This method has been compared with a modified AOAC method for the determination of total cholesterol. At a confidence level of 95%, no difference was observed between the 2 methods.     

The quantized Hall effect

Quantization of the Hall effect is one of the most surprising discoveries in recent experimental solid-state research. At low temperatures and high magnetic fields the ratio of the Hall voltage to the electric current in a two-dimensional system is quantized in units of h/e(2), where h is Planck's constant and e is the electronic charge. Concomitantly, the electrical resistance of the specimen drops to values far below the resistances of the best normal metals.