The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Volatile Fatty Acids and Hydrogen as Substrates for Sulfate-Reducing Bacteria in Anaerobic Marine Sediment

The addition of 20 mM MoO₄²⁻ (molybdate) to a reduced marine sediment completely inhibited the SO₄²⁻ reduction activity by about 50 nmol g⁻¹ h⁻¹ (wet sediment). Acetate accumulated at a constant rate of about 25 nmol g⁻¹ h⁻¹ immediately after MoO₄²⁻ addition and gave a measure of the preceding utilization rate of acetate by the SO₄²⁻-reducing bacteria. Similarly, propionate and butyrate (including isobutyrate) accumulated at constant rates of 3 to 7 and 2 to 4 nmol g⁻¹ h⁻¹, respectively. The rate of H₂ accumulation was variable, and a range of 0 to 16 nmol g⁻¹ h⁻¹ was recorded. An immediate increase of the methanogenic activity by 2 to 3 nmol g⁻¹ h⁻¹ was apparently due to a release of the competition for H₂ by the absence of SO₄²⁻ reduction. If propionate and butyrate were completely oxidized by the SO₄²⁻-reducing bacteria, the stoichiometry of the reactions would indicate that H₂, acetate, propionate, and butyrate account for 5 to 10, 40 to 50, 10 to 20, and 10 to 20%, respectively, of the electron donors for the SO₄²⁻-reducing bacteria. If the oxidations were incomplete, however, the contributions by propionate and butyrate would only be 5 to 10% each, and the acetate could account for as much as two-thirds of the SO₄²⁻ reduction. The presence of MoO₄²⁻ seemed not to affect the fermentative and methanogenic activities; an MoO₄²⁻ inhibition technique seems promising in the search for the natural substrates of SO₄²⁻ reduction in sediments.     

Characterization of <Emphasis Type="Italic">Campylobacter</Emphasis> phages including analysis of host range by selected <Emphasis Type="Italic">Campylobacter</Emphasis> Penner serotypes

Background  

The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans.     

Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology--animal welfare--has not been approached through systematic assessment and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals along various stages of post natal development. The protocol used covered reproductory performance and behaviour in GFP and wildtype sows and general health and development, social behaviour, exploratory behaviour and emotionality in GFP and wildtype littermates from birth until an age of roughly 4 months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs expressing GFP as healthy. Although the results are not surprising in the light of previous experience, they give a more solid fundament to the evaluation of GFP expression as being relatively non-invasive in pigs. The present study may furthermore serve as starting point for researchers aiming at a systematic characterization of welfare relevant effects in the line of transgenic pigs they are working with.     

Radiation from mobile phone systems: Is it perceived as a threat to people's health?

The aim of this study was to explore the prevalence, nature and determinants of concerns about mobile phone radiation. We used data from a 2006 telephone survey of 1004 people aged 15+ years in Denmark. Twenty‐eight percent of the respondents were concerned about exposure to mobile phone radiation; radiation from masts was of concern to about 15%. In contrast, 82% were concerned about pollution. Nearly half of the respondents considered the mortality risk of 3G phones and masts to be of the same order of magnitude as being struck by lightning (0.1 fatalities per million people per year) while 7% thought it was equivalent to tobacco‐induced lung cancer (～500 fatalities per million per year). Among women, concerns about mobile phone radiation were positively associated with educational attainment, perceived mobile phone mortality risk and concerns about unknown consequences of new technologies. More than two thirds of the respondents felt that they had received inadequate public information about the 3G system. The results of the study indicate that the majority of the population has little concern about mobile phone radiation while a small minority is very concerned. Bioelectromagnetics 30:393–401, 2009. © 2009 Wiley‐Liss, Inc.     

Secreted Protein Acidic and Rich in Cysteine (SPARC) in Human Skeletal Muscle

Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15–16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment. (J Histochem Cytochem 57:29–39, 2009)     

Phosphinic,phosphonic and seleninic acid bioisosteres of isonipecotic acid as novel and selective GABA(C) receptor antagonists

A number of amino acids bioisosterically derived from the specific GABA_A agonist, isonipecotic acid, were electrophysiologically characterized as antagonists at GABA_C ρ₁ receptors expressed in Xenopus oocytes. The phosphinic acid analogue of isonipecotic acid, piperidin-4-ylphosphinic acid (2), was comparable with the standard GABA_C antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), in terms of potency and GABA_C versus GABA_A receptor selectivity. Whereas the phosphonic acid analogue, piperidin-4-ylphosphonic acid (4), was at least an order of magnitude weaker than piperidin-4-ylphosphinic acid as a GABA_C antagonist, the seleninic acid analogue, piperidin-4-ylseleninic acid (SEPI, 6), was the most potent and selective GABA_C antagonist within the group of isonipecotic acid derived amino acids studied.     

Quality control in boar semen production by use of the FACSCount AF system

A simple and rapid flow cytometric method has recently been developed for simultaneous determination of sperm concentration and viability in semen from domestic animals. Use of SYBR-14 trade mark in combination with propidium iodide (PI) allows estimation of the proportion of live sperm (viability). An internal standard of fluorescent microspheres (beads) makes it possible to determine the sperm concentration during the same analysis. In the first experiment, the relationship between sperm viability and litter size was investigated. The second experiment explored whether a smaller variation in the number of motile sperm per insemination dose could be obtained using the FACSCount AF flow cytometer than using a spectrophotometer. Results in the first experiment show that sperm viability is closer related to litter size than is the traditionally used motility parameter. Although the flow cytometer is precise and objective, a limited effect on litter size should be anticipated if ejaculates are selected for insemination according to the percentage of viable sperm. However, the present trial used large insemination doses (2.3 x 10(9) motile sperm/dose) which partially compensate for the differences in motility and viability between boars and ejaculates. In the second experiment it was found that variation in the number of motile sperm per insemination dose could be reduced significantly if the FACSCount AF flow cytometer rather than the Corning 254 spectrophotometer was used for determination of sperm concentration in the raw semen. It is concluded that the FACSCount AF flow cytometer is a strong tool for improvement of the quality control in artificial insemination (AI) centres.     

Aza-THIP and related analogues of THIP as GABA C antagonists

The potency of a series of eight compounds structurally related with 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a potent GABA(A) partial agonist exhibiting GABA(C) rho(1) antagonist effect (K(i)=25 microM), was determined electrophysiologically using homomeric human GABA(C) rho(1) receptors expressed in Xenopus oocytes. Protolytic properties (pK(a) values for the acidic bioisosteric groups) and the presence of steric bulk in the molecules appear to be structural parameters of importance for blockade of the GABA(C) rho(1) receptor. Within this series of moderately potent GABA(C) antagonists, only 4,5,6,7-tetrahydropyrazolo[5,4-c]pyridin-3-ol (Aza-THIP) does not interact detectably with GABA(A) receptors, and Aza-THIP has the potential of being a useful tool for molecular and behavioural pharmacological studies.