Continuous shoot growth monitoring in hydroponics

A weighing apparatus for automatic recording of fresh weight of shoots of spinach plants ( Spinacia oleracea L., cv. Subito) growing in nutrient solution is described. The system was tested for 17 days in a controlled environment and enabled the determination of the relative growth rate (RGR) of the shoot fresh weight. Results from three consecutive growth experiments demonstrated diurnal fluctuations in the relative growth rate of the shoot fresh weight. In general, relative growth rates were between 0.32 and 0.36 day⁻¹ 16 days after sowing and decreased to between 0.11 and 0.18 day⁻¹ during the 12 following days. The variance between three replicate growth curves was compared with the variance of a growth function fitted through destructively obtained spinach shoot weight data.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO₂ of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CAP chloramphenicol     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

The cyanobacterium Oscillatoria agardhii was grown in continuous culture under various light conditions in order to study the interactions of light on phosphorus-limited growth. Under severe P-limiting (light-saturating) conditions, a low chlorophyll a and C-phycocyanin content was found. In addition, the light-harvesting capacity, reflected in the values of P _max (maximum light-saturated oxygen production rate) and (photosynthetic affinity), were low compared to light-limited cultures.Reduction of the light climate, either by reduction of the length of the photoperiod or light-intensity, resulted in an increase in light-harvesting capacity (higher pigment content, P _m and ) during growth under P-limiting conditions. Light-induced changes in P _max and could be related to the relative growth rate, being the actual growth rate as a fraction of the growth rate which would be observed under light-limiting conditions.Under P-limiting conditions, reduction of the light-climate caused a reduction in dry weight of the culture. This decrease was mainly due to a decrease in carbohydrate content of the cells. Under all conditions tested, carbohydrates were found to accumulate during the light-period and to be consumed during the dark-period.Evaluation of carbohydrate consumption in the dark yielded a specific maintenance rate constant of 0.001 h^-1. This observation leads to the conclusion that the specific maintenance rate constant is independent on the character of the growth rate limiting nutrient for O. agardhii.     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Sequential deiodination of thyroxine in rat liver homogenate.

Rat liver homogenate was incubated at 37 degrees C with thyroxine, 3,3',5-tri-iodothyronine, 3,3',5'-tri-iodothyronine or 3,3'-di-iodothyronine. The degradation or accumulation of these compounds was measured by specific radioimmunoassays. (1) Production of 3,3',5-tri-iodothyronine from thyroxine was highest at pH 6.0--6.5 and was markedly stimulated by the addition of dithiothreitol and effectively inhibited in the presence of 6-propyl-2-thiouracil. (2) Accumulation of 3,3',5'-tri-iodothyronine on incubation of thyroxine with homogenate was only observed above pH 8.5. Otherwise the product was converted into 3,3'-di-iodothyronine too rapidly to allow its measurement. By measuring 3,3'-di-iodothyronine it was deduced that 5-deiodination of thyroxine was most effective at approx. pH 8.0. Dithiothreitol powerfully stimulated this reaction and 6-propyl-2-thiouracil strongly inhibited. (3) Monodeiodination of the tyrosine ring of 3,3',5-tri-iodothyronine was the slowest reaction, was optimal at pH 8.0 and was less affected by dithiothreitol and 6-propyl-2-thiouracil than the above reactions. (4) 5'-Deiodination of 3,3',5'-tri-iodothyronine was extremely rapid, with a pH optimum probably at about 6.5. Owing to the high reaction rate under the conditions used it was not possible to assess the effects of dithiothreitol and 6-propyl-2-thiouracil.     

Genomic landscape of rat strain and substrain variation

Background

Since the completion of the rat reference genome in 2003, whole-genome sequencing data from more than 40 rat strains have become available. These data represent the broad range of strains that are used in rat research including commonly used substrains. Currently, this wealth of information cannot be used to its full extent, because the variety of different variant calling algorithms employed by different groups impairs comparison between strains. In addition, all rat whole genome sequencing studies to date used an outdated reference genome for analysis (RGSC3.4 released in 2004).

Results

Here we present a comprehensive, multi-sample and uniformly called set of genetic variants in 40 rat strains, including 19 substrains. We reanalyzed all primary data using a recent version of the rat reference assembly (RGSC5.0 released in 2012) and identified over 12 million genomic variants (SNVs, indels and structural variants) among the 40 strains. 28,318 SNVs are specific to individual substrains, which may be explained by introgression from other unsequenced strains and ongoing evolution by genetic drift. Substrain SNVs may have a larger predicted functional impact compared to older shared SNVs.

Conclusions

In summary we present a comprehensive catalog of uniformly analyzed genetic variants among 40 widely used rat inbred strains based on the RGSC5.0 assembly. This represents a valuable resource, which will facilitate rat functional genomic research. In line with previous observations, our genome-wide analyses do not show evidence for contribution of multiple ancestral founder rat subspecies to the currently used rat inbred strains, as is the case for mouse. In addition, we find that the degree of substrain variation is highly variable between strains, which is of importance for the correct interpretation of experimental data from different labs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1594-1) contains supplementary material, which is available to authorized users.     

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.