Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of¹⁴CO₂ from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Anther-specific,developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower

We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.     

Use of ars18 based vectors to increase protein production in Yarrowia lipolytica.

The isolation of ars sequence from the yeast Yarrowia lipolytica has recently been reported (Fournier et al., 1991). Vectors containing ars18 have been used to increase homologous and heterologous protein production. Examples presented are the Yarrowia lipolytica alkaline extracellular protease (AEP), the porcine alpha 1-interferon and the bovine prochymosin. A 2- to 6-fold increase in the corresponding protein production was observed and in several cases it was established that it corresponded to the copy number of plasmid in the cell.     

Computation of relaxation matrix elements from incomplete NOESY data sets

Summary The structural determination of biological molecules in solution by NMR relies on the determination of a set of interatomic distances obtained by measurement of intramolecular nuclear Overhauser effects (NOE). It is shown in this paper that it is possible to obtain the accurate relaxation rate (and hence the interatomic distance) from the direct measurement of a single NOE signal. The precise analysis of a NOESY peak evolution with respect to the mixing time allows the evaluation of the relaxation parameters for the pair of spins under consideration. This is done without any assumption on the relaxation of unmeasured spins, or on the movement of the molecule. The theoretical basis of this method is presented. In order to evaluate the proposed method, a simulated case on the protein BPTI is studied, which shows that the method performs very well even in the case of noisy data sets.     

Minimal growth factor requirements of human fetal kidney in serum- and glucose-free culture.

The addition of insulin plus transferrin to Leibovitz's L-15 medium was previously shown to restore important cellular functions in a serum-free system developed in our laboratory for human fetal kidney explants. The objective of the present study was to compare the effectiveness of this insulin plus transferrin combination with one used in other in vitro systems whereby serum is substituted by a mixture of five hormones (insulin, transferrin, hydrocortisone, triiodothyronine and prostaglandin E1). In fetal kidney it was found that the combination of insulin plus transferrin was as effective as the five-hormone mixture on DNA synthesis after 5 days of culture and was even more effective in younger fetuses (10-13 weeks) compared with older fetuses (16-19 weeks). However, protein synthesis was more sensitive to the five-hormone combination. Selective deletion of individual hormones showed that insulin is the essential factor for the growth of cultured kidney explants. Differentiation of brush border membranes in nephrons, as evaluated by alkaline phosphatase and gamma-glutamyl-transferase activities, was not significantly modified by either of the two combinations. The present results indicate that insulin plus transferrin represents the optimizing condition for our culture model. The response to supplements varies according to fetal age and possibly to tissue proliferation states and/or cell type.     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin     

Effects of direct transfer from freshwater to seawater on respiratory and circulatory variables and acid-base status in rainbow trout

Summary The effects of increased ambient salinity (35 mg · ml^-1) were studied at 1, 6, and 24 h after direct transfer of rainbow trout from freshwater to seawater. Two series of experiments were carried out successively. The first series was designed to simultaneously study all the respiratory (except Hb affinity for O₂), circulatory, and acid-base variables in each fish. In this series, fish were fitted with catheters chronically inserted into the cardiac bulbus, the dorsal aorta, and the opercular and buccal cavities. In the second series, designed to study haemoglobin O₂ affinity, fish were fitted with only a dorsal aorta catheter. The ventilatory flow ( ) was markedly increased just after transfer (by 55% at 1 h), then more moderately (by 20% at 6 h and 32% at 24 h). The initial hyperventilation peak was associated with frequent couphing motions. These ventilatory changes resulted essentially from increase in ventilatory amplitude. Initially, standard oxygen consumption (MM}O₂) decreased slightly, the moderately increased (by 12% at 24 h), so that the oxygen convection requirement ( ) increased substantially. In spite of an increased ventilation, the partial pressure of oxygen in arterial blood (P _aO₂) decreased slightly at 1 h, prior to returning to control levels, while partial pressure of carbon dioxide in arterial blood (P _aCO₂) was not significantly decreased. Gill oxygen transfer factor decreased substantially at 1 h (by 35%) then more moderately (by 7% at 1 h and 12% at 24 h). These results suggest a decrease in gas diffusing capacity of the gills. As P _aCO₂ remained approximatively unchanged, the gradual decrease in arterial pH (pH_a) from 7.94 to 7.67 at 24 h must therefore be regarded as a metabolic acidosis. The strong ion difference decreased markedly because the concentration of plasma chloride increased more than that of sodium. Arterial O₂ content (C _aO₂) gradually decreased (by 38% at 24 h) simultaneously with the decrease in pH_a, while the ratio P _aO₂/C _aO₂ increased. In parallel, seawater exposure induced a marked decrease in affinity of haemoglobin for O₂, so that at 24 h, P₅₀ was increased by 26% above the value obtained in freshwater-adapted trout. The increase in could be ascribed initially (at 1 h) to the decrease of P _aO₂ and later to a stimulation of respiratory neurons resulting from the lowered medullary interstitial pH. The decrease in C _aO₂ could be interpreted mainly as a consequence of a decreased affinity of haemoglobin for O₂, likely to be due to the blood acidosis and a predictable increase in chloride concentration within erythrocytes. Cardiac output ( ) slightly decreased at 1 h, then progressively increased by 30% at 24 h. Branchial vascular resistance increased at 1 h by 28%, then decreased by 18% of the control value at 24 h. Systemic vascular resistance decreased markedly by 40% at 24 h. As heart rate (HR) remained significantly unchanged, the cardiac stroke volume initially decreased then increased in relation to the changes in . The increase of , allowing compensation for the effect of decreased C _aO₂ in tissue O₂ supply, was interpreted as a passive consequence of the decrease in total vascular resistance occurring during seawater exposure.Abbreviations a.u. arbitrary units - C _aO₂ arterial oxygen content - pH₅₀ arterial pH at P₅₀ - C _vO₂ venous oxygen content - Hb haemoglobin - HR heart rate - Hct hematocrit - n_Hill Hill coefficient - O₂ standard oxygen consumption - P _aCO₂ arterial partial pressure of carbon dioxide - P _aO₂ arterial partial pressure of oxygen - P _vO₂ oxygen partial pressure in mixed venous blood - P₅₀ oxygen tension at half saturation of haemoglobin - P _VA, P _DA blood pressure in ventral and dorsal aorta - pH_a arterial pH - PIO₂, PEO₂ oxygen partial pressure of inspired and expired water - PO₂ oxygen partial pressure - cardiac output - SEM standard error of mean - S.I.D. strong ion difference - SV cardiac stroke volume - TO₂ gill oxygen transfer factor - U oxygen extraction coefficient - VA ventilatory amplitude - VF ventilatory frequency - VR_G, VR_S branchial and systemic vascular resistances - ventilatory flow - ventilatory oxygen convection requirement     

Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.     

Conversion of large heterologies in Streptococcus pneumoniae

In genetic transformation, long deletions dramatically increase the frequency of wild-type recombinants in 2-point crosses. In 3-point crosses in which the deletion was localized between 2 point mutations we demonstrated that this hyper-recombination was the result of genetic conversion extending over several scores of bases outside the deletion. As this conversion did not require an active DNA polymerase A gene, it was proposed that the mechanism of conversion involves breakage and ligation between DNA molecules. A similar hyper-recombination was observed when donor DNA carried an insertion. These results suggest that long heterologies participated in recombination so that surrounding homologous regions are almost completely paired and that these long heterologies are converted. It appears that it is a process that evolved to correct errors of replication which lead to long deletions and which are not eliminated by other systems.