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Biomedical subjects

P L Jørgensen

Publications and source records attributed to P L Jørgensen.

At least 19 recordsLinked to original sources
PubMed2001

Using inactivated microbial biomass as fertilizer: the fate of antibiotic resistance genes in the environment.

The waste product produced by Novo Nordisk A/S from microbial fermentations is used as agricultural fertilizer in Denmark (NovoGro) after being treated by heat and chemicals to destroy the microorganisms. The fertilizer contains DNA fragments from the genetically modified microorganisms used in industrial production. This DNA contains genes coding for the desired industrial products as well as genes used as genetic selection markers during production strain development. The antibiotic resistance markers used as genetic selection markers are chloramphenicol (Cm), kanamycin (Km) and ampicillin (Ap). The aim of the present study was to examine whether DNA and intact genes were present in NovoGro and whether horizontal transfer of DNA isolated from inactivated production strains occurred either in the laboratory or in the fields treated with NovoGro. DNA isolated from NovoGro was analysed by PCR and intact genes coding for a protease and chloramphenicol resistance were amplified. This isolated DNA was used for in vitro experiments including electroporation and transformation but no transfer of DNA to Escherichia coli or Bacillus subtilis was observed. The antibiotic resistance profile of the indigenous bacterial population in the fields treated with NovoGro compared with fields treated with inorganic fertilizers showed no differences. In addition, DNA isolated directly from the fields treated with NovoGro for up to 7 years was analysed by PCR and no specific production gene constructs could be detected.

Anti-Bacterial Agents↗
PubMed1999

Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase.

BACKGROUND: The enzymatic hydrolysis of glycosides involves the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. The covalent intermediate may be trapped on-enzyme using 2-fluoro-substituted glycosides, which provide details of the intermediate conformation and noncovalent interactions between enzyme and oligosaccharide. Xylanases are important in industrial applications - in the pulp and paper industry, pretreating wood with xylanases decreases the amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, versus glucose-based, substrates. RESULTS: The structure of the family 11 xylanase, Xyl11, from Bacillus agaradhaerens has been solved using X-ray crystallography in both native and xylobiosyl-enzyme intermediate forms at 1.78 A and 2.0 A resolution, respectively. The covalent glycosyl-enzyme intermediate has been trapped using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike covalent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual 2,5B conformation. CONCLUSIONS: The 2,5B conformation found for the alpha-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the 4C1 chair conformation observed for other systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covalent glycosyl-enzyme intermediate with the equivalent structure for the related family 12 endoglucanase, CelB, from Streptomyces lividans reveals the likely determinants for substrate specificity in this clan of glycoside hydrolases.

Bacillus↗
PubMed1996

Consequences of mutations to the phosphorylation site of the alpha-subunit of Na, K-ATPase for ATP binding and E1-E2 conformational equilibrium.

Expression of Na, K-ATPase in yeast allowed targeting of alpha beta-units with lethal substitutions at the phosphorylation site alpha 1 (D369N) beta 1 and alpha 1 (D369A) beta 1 at the cell surface at the same concentration of alpha-subunit and [3H] ouabain binding sites as for wild type Na, K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na, K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na, K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the gamma-phosphate to the free energy of ATP binding. Analysis of the K(+)-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the alpha-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H] ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD = 133 nM for wild type, and [3H] ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.

Adenosine Triphosphate↗
PubMed1996

Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding.

For understanding the regulation of sodium reabsorption, it is important to know whether the alpha 2- or alpha 3-isoform of Na-K-adenosinetriphosphatase (Na-K-ATPase) is expressed in mammalian kidney in addition to the predominant alpha 1 beta 1-isozyme. Here we applied competitive polymerase chain reaction (PCR) for estimation of mRNA in parenchymal zones of rat kidney for comparison to high-affinity [3H]ouabain binding. The alpha 3-isoform mRNA was demonstrated to form 0.04-0.05% of the amount of alpha 1-isoform mRNA in the cortex, medulla, and papilla of rat kidney. The alpha 2-mRNA was demonstrated in all three zones and constituted 0.03% of the amount of alpha 1-mRNA in cortex. The abundance of the alpha 1 truncated mRNA was 0.1-0.8% of that of the alpha 1-mRNA. The upper limit for expression of Na-K-ATPase isozyme with high ouabain affinity (dissociation constant, 69-141 nM) was 0.096-0.14% of the concentration of alpha 1 beta 1-Na-K-ATPase. Thus a small but well-defined pool of alpha 2- and alpha 3-isoforms constitutes < or = 0.1% of the amount of alpha 1-isoform at both the mRNA and protein level in rat kidney.

Animals↗
PubMed1995

A new method for integration and stable DNA amplification in poorly transformable bacilli.

We have developed a strategy for the integration and stable amplification of DNA sequences in the chromosome of poorly transformable bacilli, which avoids the presence of a functional plasmid replication system in the integrated DNA. The parental vector for integration contains two plus origins of replication from pUB110 in the same orientation on a single plasmid. Due to the direct repeats, such plasmids produce two individual progeny vectors, one of which is dependent on the other for replication, as it lacks a functional rep gene. We have used such a progeny vector system to integrate and amplify DNA on the chromosome of Bacillus licheniformis, and show that the structure is stable in the absence of selective pressure.

Bacillus↗
PubMed1995

Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis.

A deletion of the spoIIAC gene of Bacillus licheniformis was prepared in vitro by using the splicing-by-overlap-extension technique. This gene was introduced into B. licheniformis on a temperature-sensitive plasmid, and following integration and excision from the chromosome, a precisely located deletion on the chromosomal gene was prepared. The mutated bacterium was totally asporogenous and formed abortively disporic cells characterized by asymmetric septa at the poles of the cells. Qualitative plate tests revealed that the bacterium synthesized normal levels of DNase, polygalacturonate lyase, protease, RNase, and xylanase, but the hydrolysis zones due to beta-1,3-glucanase and carboxymethyl cellulase activity were smaller in the mutant than in the parent strain. The synthesis of alkaline protease was the same in batch cultures of the mutant and the parent during prolonged incubation for 72 h, but the alpha-amylase yields were reduced by about 30% by the mutation.

Amylases↗
PubMed1993

Location of Asn831 of the alpha chain of Na/K-ATPase at the cytoplasmic surface. Implication for topological models.

The principal objective of this work has been to determine whether Asn831 of the alpha chain of Na/K-ATPase is located at the cytoplasmic or extracellular surface. Asn831 is the N-terminal residue of a 19-kDa C-terminal tryptic fragment which, together with smaller (8-11 kDa) membrane-embedded fragments of the alpha chain and a largely intact beta chain, is produced by extensive tryptic digestion of renal Na,K-ATPase in the presence of Rb+ and absence of Ca2+ ions. In these so-called "19-kDa membranes," Rb+ and Na+ occlusion capacities are normal, but ATP-dependent activities are lost. Transmembrane segments in the 19-kDa and other fragments are thought to contribute ligating residues to a cation occlusion "cage." However, the arrangement of transmembrane segments of the alpha chain is uncertain, particularly in the critical C-terminal domain. Different topological models predict that the N-terminal Asn831 of the 19-kDa fragment lies at opposite surfaces, respectively. The location of Asn831 has been studied by tryptic digestion of either tight right side-out-oriented membrane vesicles isolated from renal medulla or phospholipid vesicles reconstituted with renal Na/K-ATPase, containing pumps in both right side-out and inside-out orientations. Digestion is performed in the presence of Rb+ and absence of Ca2+ ions. In native renal membrane vesicles the alpha chain is not split by trypsin. By contrast, the beta chain is partially split to a fragment of 16 kDa, beginning at the N-terminal Ala. This fragment is 4 residues longer than a 16-kDa fragment produced by digestion of purified Na/K-ATPase, a finding which shows that trypsin has full access to the extracellular surface, but not to the interior of the vesicles. In the presence of deoxycholate, which destroys the membrane permeability barrier, trypsin digests the alpha chain and the 19-kDa fragment (N-terminal Asn831) is produced. Extensive tryptic digestion of phospholipid vesicles reconstituted with the Na/K-ATPase leads to digestion of the alpha chain and appearance of intermediate fragments and then the 19-kDa fragment. About 25% of the alpha chains in the preparation are resistant to digestion. Functional studies show that the trypsin-resistant pumps are in the right side-out orientation. Observed tryptic fragments were sequenced and the following N termini were detected: Glu31, 33 kDa; Ser221, 78 kDa; Ile470, 56 kDa; Ala590, 39 kDa; and Asn831, 19 kDa. Observation of these N termini is consistent with digestion of the alpha chain of inside-out-oriented pumps at cytoplasmic sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Aldosterone and thyroid hormone modulation of alpha 1-, beta 1-mRNA, and Na,K-pump sites in rabbit distal colon epithelium. Evidence for a novel mechanism of escape from the effect of hyperaldosteronemia.

Aldosterone and thyroid hormone regulation of Na,K-pump biosynthesis has been examined in the distal colon epithelium of rabbits. Qualitative analysis of alpha-subunit isoform distribution (alpha 1, alpha 2, alpha 3) detected only the alpha 1-mRNA in the distal colon epithelium and outer renal medulla, while all three isoforms were observed in rabbit brain. A low-sodium diet led to a rise in serum aldosterone from 0.6 nM to 1.4-1.9 nM and an increase of alpha 1-mRNA to 162%, beta 1-mRNA to 120%, and the number of Na,K-pump units as determined by specific [3H]-ouabain binding to 182% of control by the second day of the diet. While aldosterone levels remained elevated, a spontaneous decrease in serum levels of T3 and T4 to 50-60% of control from the third day of the diet was followed by downregulation of beta 1-mRNA to 55-67%, alpha 1-mRNA to 63-105%, and of [3H]-ouabain binding to 103% of control, suggesting that a reduced rate of synthesis of the beta 1-subunit is rate limiting for Na,K-pump biosynthesis. Substitution with T3 (10 micrograms/kg) at the seventh day with transient restoration of serum T3 to control levels, led to rapid accumulation of beta 1-mRNA to 152%, of alpha 1-mRNA to 135%, and of the number of Na,K-pump units to 153% of control. This is consistent with thyroid hormone having a permissive role for the aldosterone stimulation of Na,K-pump biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1993

Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium.

To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca(2+)-activated K+ channels from the basolateral plasma membrane of the surface cells in the distal colon have been characterized by single channel analysis after fusion of vesicles with planar lipid bilayers. A Ca(2+)-activated K+ channel with a single channel conductance of 275 pS was predominant. The sensitivity to Ca2+ was strongly dependent on the membrane potential and on the pH. At a neutral pH, the K0.5 for Ca2+ was raised from 20 nM at a potential of 0 mV to 300 nM at -40 mV. A decrease in pH at the cytoplasmic face of the K+ channel reduced the Ca2+ sensitivity dramatically. A loss of the high sensitivity to Ca2+ was also observed after incubation with MgCl2, possibly a result of dephosphorylation of the channels by endogenous phosphatases. Modification of the channel protein may thus explain the variation in Ca2+ sensitivity between studies on K+ channels from the same tissue. High affinity inhibition (K0.5 = 10 nM) by charybdotoxin of the Ca(2+)-activated K+ channel from the extracellular face could be lifted by an outward flux of K+ through the channel. However, at the ion gradients and potentials found in the intact epithelium, charybdotoxin should be a useful tool for examination of the role of maxi K+ channels. The high sensitivity for Ca2+ and the properties of the activator site are in agreement with an important regulatory role for the high conductance K+ channel in the epithelial cells.

Animals↗
PubMed1992

celA from Bacillus lautus PL236 encodes a novel cellulose-binding endo-beta-1,4-glucanase.

celA from the cellulolytic bacterium Bacillus lautus PL236 encodes EG-A, an endo-beta-1,4-glucanase. An open reading frame of 2,100 bp preceded by a ribosome-binding site encodes a protein with a molecular mass of 76,863 Da with a typical signal sequence. The NH2-terminal active domain of EG-A is not homologous to any reported cellulase or xylanase and may represent a new family of such enzymes. A 150-amino-acid COOH-terminal peptide is homologous to noncatalytic domains in several other cellulases (A. Meinke, N.R. Gilkes, D.G. Kilburn, R.C. Miller, Jr., and R.A.J. Warren, J. Bacteriol. 173:7126-7135, 1991). Upstream of celA, a partial open reading frame encodes a 145-amino-acid peptide which also belongs to the family mentioned. Zymogram analysis of extracts from Escherichia coli and supernatants of Bacillus subtilis and B. megaterium, including protease-deficient mutants thereof, which express celA, revealed two active proteins, EG-A-L and EG-A-S, with Mrs of 74,000 and 57,000, respectively. The proportion of EG-A-L to EG-A-S depends on the extracellular proteolytic activity of the host organism, indicating that EG-A-S arises from posttranslational proteolytic modification of EG-A-L. Since EG-A-S has an NH2 terminus corresponding to the predicted NH2-terminal sequence of EG-A, processing appears to take place between the catalytic and noncatalytic domains described. EG-A-L and EG-A-S were purified to homogeneity and shown to have almost identical characteristics with respect to activity against soluble substrates and pH and temperature dependency. EG-A-L binds strongly to cellulose, in contrast to EG-A-S, and has higher activity against insoluble substrates than the latter. We conclude that the COOH-terminal 17,000-Mr peptide of EG-A-L constitutes a cellulose-binding domain.

Amino Acid Sequence↗
PubMed1992

Functional domains of Na,K-ATPase; conformational transitions in the alpha-subunit and ion occlusion.

The purified membrane-bound Na,K-ATPase from the outer renal medulla crystallizes in different forms with monomeric or dimeric unit cells, but the alpha beta-unit is the minimum asymmetric unit in P1-crystals, and cation binding sites and occlusion cavities for Na+ or K+(Rb+) are formed within the structure of the alpha beta-unit. Domains have been identified that engage in structural transitions mediating energy transduction between ATP or phosphorylation sites in the cytoplasmic protrusion and cation sites in the membrane. The purpose of this study has been to examine the relationship between ion binding and occlusion, and transitions between two conformations of the alpha-subunit, the Na-form (E1) or the K-form (E2) that are defined by the kinetics of inactivation of Na,K-ATPase and K-phosphatase activities and a specific sequence of tryptic (E1: T2(Lys30)-T3(Arg262); E2: T1(Arg438)-T2(Lys30)) or chymotryptic (E1: C3(Leu266)) cleavage of the alpha-subunit. In E1-forms stabilized in an oligomycin complex, three sites for Na+ occlusion were found per alpha-subunit. In E2-forms stabilized as the phosphorylated ouabain complex, two Na+ or two Rb+ were occluded per alpha-subunit, the E2[2Rb] conformation having a higher affinity. Selective chymotryptic cleavage (Leu266) in the second cytoplasmic loop completely blocks E1-E2 transition, ion- and charge translocation without affecting ligand binding to the protein. Structural transitions of this segment reflect changes in capacity and orientation of cation sites that are part of the ion translocation process in the alpha-subunit of the Na,K-pump.

Animals↗
PubMed1991

Cloning of a chromosomal alpha-amylase gene from Bacillus stearothermophilus.

We have cloned and sequenced a gene for a heat-stable alpha-amylase from a natural isolate of Bacillus stearothermophilus. Previously, it had been shown that B. stearothermophilus amylase genes may be harboured on indigenous plasmids. We have found that our isolate harbours the amylase gene only on the chromosome and not on its indigenous plasmid.

Amino Acid Sequence↗