Biomedical subjects

Dan Nilsson

Publications and source records attributed to Dan Nilsson.

11 recordsLinked to original sources

PubMed2004

Using spectrophotometry to determine in vitro turnover rates of peptides in plasma.

A new method for determination of first-order elimination constants for dipeptides is presented. The peptides are hydrolysed by plasma enzymes into amino acids, and ortho-phthaldialdehyde (OPA) is used to react with free primary amino groups. The concentration of free amino groups can, thus, be followed using simple spectrophotometry. A mathematical model for the concentration of free primary amino groups with time is presented through which the elimination constant, and thus the half life, can be determined by curve fitting. The method is applied to inhibitors of angiotensin-converting enzyme derived from the primary structure of milk proteins. The results show that these dipeptides have in vitro half lives ranging from 4.3-64 min, when incubated with 50% rat plasma. This explains why these casokinins in vivo only cause a very moderate and short-lasting inhibition. The model for calculation of elimination constant is limited to dipeptides that do not contain a C-terminal proline. The derivatization method can be applied to longer peptides as a crude indicator of peptide hydrolysis, but does not allow calculation of their elimination constants per se.

Animals↗

PubMed2003

A simple visual system without neurons in jellyfish larvae.

Earlier detailed studies of cnidarian planula larvae have revealed a simple nervous system but no eyes or identifiable light sensing structures. Here, we describe the planula of a box jellyfish, Tripedalia cystophora, and report that these larvae have an extremely simple organization with no nervous system at all. Their only advanced feature is the presence of 10-15 pigment-cup ocelli, evenly spaced across the posterior half of the larval ectoderm. The ocelli are single cell structures containing a cup of screening pigment filled with presumably photosensory microvilli. These rhabdomeric photoreceptors have no neural connections to any other cells, but each has a well-developed motor-cilium, appearing to be the only means by which light can control the behaviour of the larva. The ocelli are thus self-contained sensory-motor entities, making a nervous system superfluous.

Animals↗

PubMed2003

Lactic acid bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo.

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 microg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 microg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II.

Angiotensin I↗

PubMed2003

Characterization of new milk-derived inhibitors of angiotensin converting enzyme in vitro and in vivo.

Inhibition of angiotensin converting enzyme (ACE) has been observed with a variety of different peptides, and peptide fragments with inhibitory capabilities have been identified within many different proteins, including milk proteins. The purpose of this study therefore was to identify new short peptides with inhibitory properties from the primary structure of milk proteins and to characterize them in vitro and in vivo, since no milk derived ACE inhibitors have previously been evaluated for their ability to inhibit ACE in vivo. In vitro, 8 of 9 dipeptides were found to be competitive inhibitors of ACE. The IC50 was significantly lower when an angiotensin I-like substrate was used, than when a bradykinin-like substrate was used. Using three different in vivo models for ACE inhibition, a very moderate effect was observed for three of the new peptides, but only for up to 6 or 12 minutes. Nothing was observed with two reference compounds that are reported to be hypotensive ACE-inhibitors derived from milk proteins. This raises the question whether the mechanism of hypotensive action is straightforward inhibition of ACE in vivo.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides.

A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides.

Acetoin Dehydrogenase↗

PubMed2002

The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth.

Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells.

Adenosine Diphosphate↗

PubMed2002

Increasing acidification of nonreplicating Lactococcus lactis deltathyA mutants by incorporating ATPase activity.

Lactococcus lactis MBP71 deltathyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.

Adenosine Triphosphatases↗

PubMed2002

Bacteriophage resistance of a deltathyA mutant of Lactococcus lactis blocked in DNA replication.

The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 deltathyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.

Bacteriophages↗

PubMed2002

Cardiovascular effects of fermented milk containing angiotensin-converting enzyme inhibitors evaluated in permanently catheterized, spontaneously hypertensive rats.

In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2002

Expression of genes encoding F(1)-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis.

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F(1) domain of the membrane-bound (F(1)F(0)) H(+)-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F(1)-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F(1)-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.

Adenosine Triphosphatases↗

PubMed2002

The glycolytic flux in Escherichia coli is controlled by the demand for ATP.

The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F(1) part of the membrane-bound (F(1)F(0)) H(+)-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly "carbon" limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.

Adenosine Triphosphate↗