Indirect RT-PCR in-situ hybridization: a novel non-radioactive method for detecting glucose-dependent insulinotropic peptide

To establish indirect in-situ PCR for the detection of intestinal peptide hormones, rat intestine and a murine intestinal tumor cell line, STC 1, were used. The results exhibited intensive staining of GIP-producing K-cells. Paraformaldehyde-fixed cryostat sections yielded the best results in signal to background ratio with RT-PCR in-situ hybridization. Moreover, it was possible to elevate the positive staining signal and to reduce background staining. Digoxigenin-labeled in-situ hybridization served as a control for specificity and sensitivity of GIP (glucose-dependent insulinotropic peptide) mRNA expression on cryostat as well as paraffin sections. In conclusion, this RT-PCR in-situ hybridization protocol proves to be a specific, sensitive and reliable non-radioactive technique for the detection of intestinal peptide hormone mRNA, especially in tissues or tumor cells where the application of ISH is limited.     

Approaches towards understanding methionine biosynthesis in higher plants

Summary. Plants are able to synthesise all amino acids essential for human and animal nutrition. Because the concentrations of some of these dietary constituents, especially methionine, lysine, and threonine, are often low in edible plant sources, research is being performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their transport, synthesis and accumulation in plants. This knowledge can be used to develop strategies allowing a manipulation of crop plants, eventually improving their nutritional quality. This article is intended to serve two purposes. The first is to provide a brief review on the physiology of methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of methionine in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth. This information can be used to develop strategies to improve methionine content of plants and to provide crops with a higher nutritional value. Received January 28, 2000 Accepted March 3, 2000     

Manipulation of thiol contents in plants

As sulfur constitutes one of the macronutrients necessary for the plant life cycle, sulfur uptake and assimilation in higher plants is one of the crucial factors determining plant growth and vigour, crop yield and even resistance to pests and stresses. Inorganic sulfate is mostly taken up as sulfate from the soil through the root system or to a lesser extent as volatile sulfur compounds from the air. In a cascade of enzymatic steps inorganic sulfur is converted to the nutritionally important sulfur-containing amino acids cysteine and methionine (Hell, 1997; Hell and Rennenberg, 1998; Saito, 1999). Sulfate uptake and allocation between plant organs or within the cell is mediated by specific transporters localised in plant membranes. Several functionally different sulfate transporters have to be postulated and have been already cloned from a number of plant species (Clarkson et al., 1993; Hawkesford and Smith, 1997; Takahashi et al., 1997; Yamaguchi, 1997). Following import into the plant and transport to the final site of reduction, the plastid, the chemically relatively inert sulfate molecule is activated through binding to ATP forming adenosine-5'-phosphosulfate (APS). This enzymatic step is controlled through the enzyme ATP-sulfurylase (ATP-S). APS can be further phosphorylated to form 3'-phosphoadenosine-5'-phosphosulfate (PAPS) which serves as sulfate donor for the formation of sulfate esters such as the biosynthesis of sulfolipids (Schmidt and J?ger, 1992). However, most of the APS is reduced to sulfide through the enzymes APS-reductase (APR) and sulfite reductase (SIR). The carbon backbone of cysteine is provided through serine, thus directly coupling photosynthetic processes and nitrogen metabolism to sulfur assimilation. L-serine is activated by serine acetyltransferase (SAT) through the transfer to an acetyl-group from acetyl coenzyme A to form O-acetyl-L-serine (OAS) which is then sulhydrylated using sulfide through the enzyme O-acetyl-L-serine thiol lyase (OAS-TL) forming cysteine. Cysteine is the central precursor of all organic molecules containing reduced sulfur ranging from the amino acid methionine to peptides as glutathione or phytochelatines, proteines, vitamines, cofactors as SAM and hormones. Cysteine and derived metabolites display essential roles within plant metabolism such as protein stabilisation through disulfide bridges, stress tolerance to active oxygen species and metals, cofactors for enzymatic reactions as e.g. SAM as major methylgroup donor and plant development and signalling through the volatile hormone ethylene. Cysteine and other metabolites carrying free sulfhydryl groups are commonly termed thioles (confer Fig. 1). The physiological control of the sulfate reduction pathway in higher plants is still not completely understood in all details. The objective of this paper is to summarise the available data on the molecular analysis and control of cysteine biosynthesis in plants, and to discuss potentials for manipulating the pathway using transgenic approaches.     

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO(2) from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5'-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO(2) led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO(2) on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO(2), APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO(2) also recovered both enzyme activities, with OAS again influenced only APR. (35)SO(4)(2-) feeding showed that treatment in air without CO(2) severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of (35)S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of (35)S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.     

Hydroxylated 2-amino-3H-phenoxazin-3-one derivatives as products of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) biotransformation by Chaetosphaeria sp., an endophytic fungus from Aphelandra tetragona

The biotransformation of the phytoanticipin HBOA and its major degradation metabolites 2-hydroxy-N-(2-hydroxyphenyl)acetamide (7) and N-(2-hydroxyphenyl)acetamide (8) by Chaetosphaeria sp., an endophytic fungus isolated from Aphelandra tetragona, was studied. Three new metabolites could be identified as 2-amino-7-hydroxy-3H-phenoxazin-3-one (12), 2-acetylamino-7-hydroxy-3H-phenoxazin-3-one (13) and 7-hydroxy-2-(2-hydroxyacetyl)-amino-3H-phenoxazin-3-one (14). Structure elucidation of 12 and 13 was performed by MS, 1H, 13C NMR and 2D NMR techniques and confirmed by chemical transformation.     

A shoot meristem-like organ in animals; monopodial and sympodial growth in Hydrozoa

Thecate Hydrozoa produce stems from which polyps branch off. Similar to plants these stems form in two ways, either in a sympodial or in a monopodial type of growth. In the latter group a terminal organ develops which has similarities to a shoot apical meristem of higher plants: it elongates without a further differentiation. Similar to leaf formation in plants, thecate Hydrozoa produce polyps in a repetitive manner. This process continues during the whole life of the animal and has not yet been found to be limited by internal mechanisms. We studied the monopodially growing thecate Hydrozoon Dynamena pumila and suggest that the stem tip, the apical shoot meristem-like organ, is a polyp primordium hindered to develop into a polyp by the laterally developing polyps.     

Artifact production in the assay of anhydroecgonine methyl ester in serum using gas chromatography-mass spectrometry

The detection of the pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) after cocaine smoking using gas chromatography-mass spectrometry is hampered by the artifactual production of AEME. The amount of AEME increases with the amount of cocaine used producing false positive values in authentic samples. A method for the correction of quantitative values was established using calibration of pyrolysis and estimation of the artifactual AEME. Authentic AEME in serum was differentiated from the artifact above 3.5 microg/l, 99% prediction limits of the quantitation were +/-3.1 microg/l. In 16 serum samples and five postmortem blood samples, cocaine and AEME were detected, but after application of the correction method only ten were truly positive for AEME.     

Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.