Characterization of the Japanese pufferfish (Takifugu rubripes) T-cell receptor α locus reveals a unique genomic organization

Polymerase chain reactions with degenerate V gene segment primers were used to isolate the putative T-cell receptor alpha-chain gene (TCRA) from Japanese pufferfish (Takifugu rubripes). The putative TCRA chain cDNA is composed of an N-terminus leader peptide followed by the variable region and the constant region. The variable portion of the TCRA gene is encoded by V and J gene segments separated in the germline. As in mammals, the V-J junction sequences are GC rich and highly diversified. Amino acid residues that are required to maintain the function and structural integrity of the TCRA polypeptide, including the conserved Trp-Tyr-Lys and Tyr-Tyr-Cys motifs in the V gene segments, the Lys-Leu-X-Phe-Gly-X-Gly-Thr-X-Leu motif in the J gene segment, the three cysteine residues in the constant region and the charged residues in the transmembrane region are all preserved in the pufferfish. These conserved features suggest that the TCRA gene families in fish and mammals have evolved from a common ancestor.     

Sensitive characterization of microbial ubiquinones from biofilms by electrospray/mass spectrometry

Utilizing high-performance liquid chromatography/electrospray/tandem mass spectrometric analysis of the neutral lipid extract of microbial cells and biofilm communities, respiratory ubiquinone (UQ) (1-methyl-2-isoprenyl-3,4-dimethoxyparabenzoquinone) isoprenologues can be separated isocratically in minutes and assayed with a limit of quantification (LOQ) of 9 p.p.b. (11.1 fmol UQ₉ µL⁻¹). This corresponds to about 1.29 × 10⁷ cells of Pseudomonas putida . Highest sensitivity is achieved using flow-injection analysis with multiple reaction monitoring wherein ammoniated molecular ions of specific isoprenologues pass through quadrupole one, are collisionally dissociated in quadrupole two and identified from the product ion fragment at m/z 197.1 in quadrupole three. This assay has a repeatability of between 6% and 10% over three orders of magnitude ( r ² = 0.996). Quinone profiling based on dominant isoprenologue patterns provides taxonomic insights. Detection of prominent UQ isoprenologues indicates presence of microeukaryotes and α Proteobacteria with UQ₁₀, obligatory aerobic Gram-negative bacteria with UQ_4-14, facultative Gram-negative (and some γ Proteobacteria growing in microniches with oxygen or to a much lesser extent nitrate as a terminal electron acceptor with UQ_8, and other γ Proteobacteria with UQ₉. High sensitivity is essential as the phospholipid fatty acid (PLFA) to UQ molar ratios are 130 or greater. Previous studies have established that recovery of sediment communities with high PLFA/UQ ratios corresponded to areas of aerobic metabolism, an important consideration in bioremediation or nuclide mobilization.     

Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system

The environmental saphrophyte Burkholderia pseudomallei is the causative agent of melioidosis, a systemic, potentially life-threatening condition endemic to many parts of south-east Asia and northern Australia. We have used the soil nematode Caenorhabditis elegans as a model host to characterize the mechanisms by which this bacterium mounts a successful infection. We find that C. elegans is susceptible to a broad range of Burkholderia species, and that the virulence mechanisms used by this pathogen to kill nematodes may be similar to those used to infect mammals. We also find that the specific dynamics of the C. elegans-B. pseudomallei host-pathogen interaction can be highly influenced by environmental factors, and that nematode killing results at least in part from the presence of a diffusible toxin. Finally, by screening for bacterial mutants attenuated in their ability to kill C. elegans, we genetically identify several new potential virulence factors in B. pseudomallei. The use of C. elegans as a model host should greatly facilitate future investigations into how B. pseudomallei can interact with host organisms.     

Ecosystem function for water retention and forest ecosystem conservation in a watershed of the Yangtze River

The ecosystem function for water retention in a watershed of the Yangtze River was discussed in this study. The watershed was divided into 90 types of vegetation–soil–slope complex. A GIS-embodied spatial database was used to explore the relationships between the capacity of water retention by a complex and its types of vegetation, soil and slope. Furthermore, the capacity of water retention of every complex was estimated statistically by using estimation models. The spatial distribution of various capacity of water retention in the watershed was demarcated on a map based on the attributions and the locations of complexes. In addition, we evaluated integrally the situation of water retention in the watershed based on the estimation for the complexes from which it was distinctly recognized that the serious situation mainly results from the poor capacity of water retention of vegetation. The variation-location effect describes the phenomenon in which an identical variation of a factor may produce different effects on overall situation, when this variation occurs in a different spatial location. According to the variation-location effect on the expansion of forestland, a strategy 'spatial pattern-based forestland extension was proposed to conserve forest ecosystem and improve the situation of water retention in the watershed.     

Computer-integrated finite element modeling of human middle ear

 The objective of this study was to produce an improved finite element (FE) model of the human middle ear and to compare the model with human data. We began with a systematic and accurate geometric modeling technique for reconstructing the middle ear from serial sections of a freshly frozen temporal bone. A geometric model of a human middle ear was constructed in a computer-aided design (CAD) environment with particular attention to geometry and microanatomy. Using the geometric model, a working FE model of the human middle ear was created using previously published material properties of middle ear components. This working FE model was finalized by a cross-calibration technique, comparing its predicted stapes footplate displacements with laser Doppler interferometry measurements from fresh temporal bones. The final FE model was shown to be reasonable in predicting the ossicular mechanics of the human middle ear. Received: 18 February 2002 / Accepted: 6 June 2002 The preparation of temporal bone histological sections of Robert K. Dyer, Jr., MD is gratefully recognized. The Whitaker Foundation supported this work (Research Grant RG-98-0305).     

Isolation and characterization of the CYP71D16 trichome-specific promoter from Nicotiana tabacum L

Trichomes are specialized epidermal cells that produce secretions that are thought to provide a first line of defence against pests and pathogens. Many trichome-secreted compounds are used commercially as flavourings, medicines, etc. Described here is the cloning and characterization of the promoter of a tobacco trichome-specific P450 gene, CYP71D16. This promoter is shown to direct the specific expression of the reporter gene, beta-glucuronidase (GUS), in glandular trichomes of Nicotiana tabacum cv. T.I. 1068 at all developmental stages. With the full promoter, GUS activity was predominantly in the gland cell, with less in the stalk cell adjacent to the gland, and in lower stalk cells. GUS staining was also observed in the most distal trichome stalk cells of non-glandular trichomes found on variety T.I. 1112. Promoter deletion analysis revealed that the region from -223 to +111 bp is sufficient to direct trichome-specific expression, but not strong gland expression. Examination of the literature suggests that this is the first characterized trichome-specific-promoter shown to function at all stages of plant development. This promoter may provide efficient bioengineering to enhance pest and pathogen resistance, and for molecular farming based on the trichome gland system.     

On the progressive enrichment of the oxygen isotopic composition of water along a leaf

A model has been derived for the enrichment of heavy isotopes of water in leaves, including progressive enrichment along the leaf. In the model, lighter water is preferentially transpired leaving heavier water to diffuse back into the xylem and be carried further along the leaf. For this pattern to be pronounced, the ratio of advection to diffusion (Péclet number) has to be large in the longitudinal direction, and small in the radial direction. The progressive enrichment along the xylem is less than that occurring at the sites of evaporation in the mesophyll, depending on the isolation afforded by the radial Péclet number. There is an upper bound on enrichment, and effects of ground tissue associated with major veins are included. When transpiration rate is spatially nonuniform, averaging of enrichment occurs more naturally with transpiration weighting than with area-based weighting. This gives zero average enrichment of transpired water, the modified Craig-Gordon equation for average enrichment at the sites of evaporation and the Farquhar and Lloyd (In Stable Isotopes and Plant Carbon-Water Relations, pp. 47-70. Academic Press, New York, USA, 1993) prediction for mesophyll water. Earlier results on the isotopic composition of evolved oxygen and of retro-diffused carbon dioxide are preserved if these processes vary in parallel with transpiration rate. Parallel variation should be indicated approximately by uniform carbon isotope discrimination across the leaf.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

Rapid labeling of neuronal populations by ballistic delivery of fluorescent dyes

Particle-mediated ballistic delivery of fluorescent dyes has been recently used to label neuronal populations in a rapid and efficient fashion. Here we describe detailed protocols for this technique as well as recent improvements in its implementation. This technique allows rapid labeling of entire neurons in a Golgi-like manner after membranes of individual neurons are contacted by particles coated with lipophilic dyes. Neurons can be labeled by dyes of different colors at controlled densities to facilitate the study of structural interactions between cells. Furthermore, in conjunction with other histochemical labeling methods, the technique can be used to study changes in neuronal structures associated with pathologic processes in animal models or postmortem human brain. In addition to lipophilic dyes, water-soluble molecules such as calcium indicators can also be delivered efficiently with this technique. The method of ballistic delivery of indicators thus provides new avenues to probe the structure and function of the nervous system.     

Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking

Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1(-/-)). HIP1(-/-) mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1(-/-) mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis.