Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.     

Expression of PSACH-associated mutant COMP in tendon fibroblasts leads to increased apoptotic cell death irrespective of the secretory characteristics of mutant COMP.

OBJECTIVE: Pseudoachondroplasia (PSACH) is a dominantly inherited chondrodysplasia associated with mutations of cartilage oligomeric matrix protein (COMP), characterized clinically by disproportionate dwarfism and laxity of joints and ligaments. Studies in chondrocytes and cartilage biopsies suggest that the cartilage disease is caused by retention of mutant COMP in the endoplasmic reticulum of chondrocytes and by disruption of the collagen network of the extracellular matrix. The pathogenesis of the tendon disease remains unclear in the absence of a cell culture model, with available tendon biopsies leading to conflicting results with respect to the intracellular retention of mutant COMP. METHODS: We established a cell culture model using adenoviral gene transfer in tendon fibroblast cultures. We compared the effect of expression of three PSACH-associated COMP mutants and the wildtype protein on COMP secretion, matrix composition and cellular viability. RESULTS: Our results show that mutants D475N and D469Delta are retained within the endoplasmic reticulum of tendon cells similar to what is known from chondrocytes, whereas mutant H587R is secreted like wildtype COMP. In spite of this difference, the collagen I matrix formed in culture appears disturbed for all three mutants. All COMP-mutants induce apoptotic cell death irrespective of their differing secretion patterns. CONCLUSION: Pathogenic pathways leading to tendon disease in humans appear to be heterogeneous between different COMP mutants.     

Growth characteristics of microorganisms on commercially available animal-free alternatives to tryptic soy medium

The growth characteristics of a range of bacteria and fungi that are routinely used in quality control practices were compared for two representative vegetable-based tryptic soy formulations. All of the representative microorganisms grew well on the vegetable-based media and the media provided suitable recoveries of the organisms following simulated storage. Subtle phenotypic changes were observed between cultures grown on different media, but these did not significantly change the strain identification.     

A novel approach to sequence validating protein expression clones with automated decision making

Background  

Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation.     

The millipede family Polydesmidae in Taiwan, with descriptions of five new species (Polydesmida, Diplopoda)

Polydesmidae are represented in Taiwan by seven species in two genera. Neither of the genera is endemic to Taiwan, but six of the species are, including five new: Nipponesmus minorsp. n., Epanerchodus bispinosussp. n., Epanerchodus curtigonopussp. n., Epanerchodus flagellifersp. n. and Epanerchodus pinguissp. n. In addition, the diagnosis of the hitherto enigmatic genus Nipponesmus Chamberlin & Wang, 1953 is refined vis-à-vis the especially similar, Central Asian, Siberian and Eastern European genus Schizoturanius Verhoeff, 1931, chiefly based on new material of the type-species Nipponesmus shirinensis Chamberlin & Wang, 1953; this species is adequately redescribed and represents still another Taiwanese endemic. A key to all three currently known species of Nipponesmus Chamberlin & Wang, 1953 is given. The highly speciose Central to East Asian genus Epanerchodus Attems, 1901 is represented in Taiwan by five species, all keyed, including Epanerchodus orientalis Attems, 1901, which is long known to be highly variable in Japan and found particularly polymorphous and apparently allochthonous in Taiwan. The following synonymy is formalized: Epanerchodus orientalis orientalis Attems, 1901 = Epanerchodus orientalis takakuwai Verhoeff, 1913, syn. n. The genus Usbekodesmus Lohmander, 1932 is formally synonymized with Epanerchodus Attems, 1901, syn. n., resulting in the following new formal transfers: Epanerchodus redikorzevi (Lohmander, 1932), Epanerchodus swatensis (Golovatch, 1991), Epanerchodus varius (Geoffroy & Golovatch, 2004), Epanerchodus anachoretus (Golovatch, 1986), Epanerchodus buddhis (Golovatch, 1986), Epanerchodus occultus (Golovatch, 1986), Epanerchodus sacer (Golovatch, 1987), Epanerchodus theocraticus (Golovatch, 1990) and Epanerchodus theosophicus (Golovatch, 1986), all comb. n. ex Usbekodesmus. The distributions of all seven species of Polydesmidae occurring in Taiwan are mapped and discussed.     

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene – pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour. Received: 27 April 1998; in revised form: 5 August 1998 / Accepted: 11 August 1998     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium     

Trophic niche overlap and wild ungulate consumption by red fox and wolf in a mountain area in Italy

Red fox (Vulpes vulpes) and wolf (Canis lupus) are two widespread opportunistic predators living in simpatry in many areas. Nonetheless, scarce information are available on their trophic interactions. We investigated food habits of these two carnivores in a mountain area in Italy and assessed the extent of their trophic niche overlap, focusing on the consumption of wild ungulates. Thereby we analyzed the content of 669 red fox scats and 253 wolf scats collected between May 2008 and April 2009. Red foxes resulted to have a more than three times higher niche breadth than wolves. Vegetables, small mammals, wild ungulates, and invertebrates were major items (altogether 92% of volume) of the red fox annual diet. On the contrary wolf annual diet relied on wild ungulates (94% of volume) with wild boar (Sus scrofa) being the main food item. The degree of trophic niche overlap between the two species was found to be low (Pianka's O = 0.356). Diet variation between the warm and the cold seasons was limited in both species, and higher in red fox than in wolf. The two canids appeared to use wild ungulates unevenly being the former more selective for younger preys, smaller in size (newborn piglets and roe deer Capreolus capreolus fawns), whereas the latter exhibited a preference for medium-sized and large ungulates (10–35 kg wild boar and adult roe deer). Even if wild ungulates represent the main shared food category, the different use of age/weight classes by the two predators, together with their possible consumption as carrions by red fox, suggests a very limited trophic competition between wolf and red fox.This study represents a contribution to the knowledge of trophic interaction in predator–prey systems where sympatric carnivores are present.     

Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells

The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).