Biomedical subjects

S B Petersen

Publications and source records attributed to S B Petersen.

At least 37 records · Page 2Linked to original sources

PubMed1997

Radiation-induced changes in phosphorus T1 values in human melanoma xenografts studied by 31P-MRS.

31P-magnetic resonance spectroscopy (MRS) has been shown to be a promising method for monitoring tumor response to radiation therapy. The purpose of the work reported here was to investigate whether the usefulness of 31P-MRS might be enhanced by measurement of spin-lattice relaxation times (T1s) in addition to resonance ratios. The work was based on the hypothesis that tumors having a high probability of being controlled locally would show shortened T1s during the treatment course due to reoxygenation and development of necrosis. BEX-t human melanoma xenografts, which show efficient reoxygenation and development of necrosis following single dose irradiation, were used as tumor models. Tumors were treated with single doses of 5.0 or 15.0 Gy and the T1s of the inorganic phosphate and nucleoside triphosphate beta resonances were measured as a function of time after irradiation by using the superfast inversion recovery method. Fractional tumor water content was determined by drying excised tumors at 50 degrees C until a constant weight was reached. The T1s in irradiated tumors were either longer than or not significantly different from those in unirradiated control tumors. The increase in the T1s following irradiation coincided in time with a radiation-induced increase in tumor water content, suggesting a causal relationship. The effects of reoxygenation and development of necrosis on T1s were probably overshadowed by the effects of tumor water content. Consequently, the usefulness of 31P-MRS in monitoring tumor response to radiation therapy might not be significantly enhanced by measurement of T1s.

Animals↗

PubMed1997

BBReader: a computer program for the combined use of the BioMagResBank and PDB databases.

A computer program ('BBReader') was developed which performs an inverse search in the BioMagResBank database. Given (cross) peak positions of a protein, the program searches for atoms with matching chemical shifts and suggests possible assignments for user-specified homo- and heteronuclear one- to three-dimensional COSY- and NOESY-type experiments. It can handle 1H, 13C and 15N spectra. Distance information from PDB files can be utilized for filtering possible NOESY cross peak assignments.

Databases, Factual↗

PubMed1997

Simulation of protein conformational freedom as a function of pH: constant-pH molecular dynamics using implicit titration.

Solution pH is a determinant parameter on protein function and stability, and its inclusion in molecular dynamics simulations is attractive for studies at the molecular level. Current molecular dynamics simulations can consider pH only in a very limited way, through a somewhat arbitrary choice of a set of fixed charges on the titrable sites. Conversely, continuum electrostatic methods that explicitly treat pH effects assume a single protein conformation whose choice is not clearly defined. In this paper we describe a general method that combines both titration and conformational freedom. The method is based on a potential of mean force for implicit titration and combines both usual molecular dynamics and pH-dependent calculations based on continuum methods. A simple implementation of the method, using a mean field approximation, is presented and applied to the bovine pancreatic trypsin inhibitor. We believe that this constant-pH molecular dynamics method, by correctly sampling both charges and conformation, can become a valuable help in the understanding of the dependence of protein function and stability on pH.

Aprotinin↗

PubMed1995

13C NMR study of IGF-I- and insulin-effects on mitochondrial function in cultured brain cells.

Development of differential cell culture systems has facilitated the study of cellular and physiological actions of growth factors. Here we report the novel effects of insulin and insulin-like growth factor (IGF)-I on energy metabolism in cerebral cortical neurones, astrocytes and co-cultures thereof. Incorporation of label from [1-13C]glucose into lactate has been shown to involve both glycolysis and the tricarboxylic acid cycle in astrocytes. Thus 13C nuclear magnetic resonance spectroscopy was used to monitor mitochondrial function by measuring 13C labelling of the C-2 position of lactate. It could be demonstrated that insulin and IGF-I have similar effects on mitochondria but individual effects on cytosolic glucose metabolism. Evidence is presented that IGF-I and insulin stimulate neurones to release factors affecting astrocytic lactate production. Furthermore, lactate released from neuronal-astrocytic co-cultures was mostly of astrocytic origin, implicating the importance of lactate for neuronal metabolism.

Animals↗

PubMed1995

Glutamate and glutamine metabolism in cultured GABAergic neurons studied by 13C NMR spectroscopy may indicate compartmentation and mitochondrial heterogeneity.

Primary cultures of mouse cerebral cortical neurons were incubated for 3 h with 250 microM [U-13C]glutamate or 250 microM [U-13C]glutamine and 6 mM glucose. 13C NMR spectra of cell extracts exhibited distinct multiplets for glutamate, aspartate and GABA. Incorporation of label into aspartate can only occur through the tricarboxylic acid (TCA) cycle, but as demonstrated by formation of the 3,4-13C2-isotopomer of GABA and the 1,2,3-13C3-isotopomer of glutamate, these amino acids were also to some extent derived from this pathway. Formation of the 1,2-13C2-(1J1,2 = 50 Hz) and 3,4-13C2-isotopomer (1J3,4 = 50.9 Hz) in aspartate occurs exclusively when oxaloacetate (containing 12C) is derived from the second turn of the TCA cycle. From [U-13C]glutamine, the 12C containing isotopomers in aspartate accounted for 27% of the total label and from [U-13C]glutamate, it was less than 10%. When [U-13C]glutamine was the precursor, 36% of the labeled glutamate and 52% of the labeled GABA contained 12C incorporated during the first turn of the TCA cycle. These numbers decreased to 15 and 30%, respectively when [U-13C]glutamate was used. Since glutamine must be converted to glutamate before it can enter the TCA cycle as 2-oxoglutarate, appearance of 12C incorporation in aspartate should be identical when [U-13C]glutamate and [U-13C]glutamine was used as substrates. This was, however, not observed which may be indicative of (1) compartmentation of mitochondrial glutamate metabolism or (2) differences in the amount of intramitochondrial glutamate derived from external glutamate or glutamine.

Animals↗

PubMed1995

Effects of aspartame on 45Ca influx and LDH leakage from nerve cells in culture.

Aspartame (ASM), an artificial sweetener, was shown to dose dependently increase 45Ca-influx into and lactate dehydrogenase (LDH) leakage from murine brain cell cultures. Astrocytes were more resistant than neurones to the effects of ASM. In cerebellar granule neurones, a 20% increase in calcium was found after an incubation time of 22 h in the presence of 0.1 mM ASM; at 0.5 mM concentration, calcium influx increased 40% compared with control cultures. At a concentration of 10 mM, influx was increased 13-fold after 5 h. Morphological appearance as judged by phase contrast microscopy was first visibly affected after exposure to 1 mM ASM for 22 h. Citrate, another food additive, was included in the study to demonstrate that cerebellar granule neurones could tolerate 10 mM additions to the medium and citrate did not cause 45Ca influx or morphological changes in neurones after 22 h. LDH leakage, a sign of severe cell damage, was observed at 1 mM concentrations of ASM after 22 h. Cerebral astrocytes on the other hand were more resistant and showed morphological changes, increased calcium influx and LDH leakage first at 5 mM concentrations of ASM.

Animals↗

PubMed1995

31P NMR spectroscopy studies of phospholipid metabolism in human melanoma xenograft lines differing in rate of tumour cell proliferation.

The concentration of phospholipid metabolites in tumours has been hypothesized to be related to rate of cell membrane turnover and may reflect rate of cell proliferation. The purpose of the study reported here was to investigate whether 31P NMR resonance ratios involving the phosphomonoester (PME) or phosphodiester (PDE) resonance are correlated to fraction of cells in S-phase or volume-doubling time in experimental tumours. Four human melanoma xenograft lines (BEX-t, HUX-t, SAX-t, WIX-t) were included in the study. The tumours were grown subcutaneously in male BALB/c-nu/nu mice. 31P NMR spectroscopy was performed at a magnetic field strength of 4.7 T. Fraction of cells in S-phase was measured by flow cytometry. Tumour volume-doubling time was determined by Gompertzian analysis of volumetric growth data. BEX-t and SAX-t tumours differed in fraction of cells in S-phase and volume-doubling time, but showed similar 31P NMR resonance ratios. BEX-t and WIX-t tumours showed significantly different 31P NMR resonance ratios but similar fractions of cells in S-phase. The 31P NMR resonance ratios were significantly different for small and large HUX-t tumours even though fraction of cells in S-phase and volume-doubling time did not differ with tumour volume. None of the 31P NMR resonance ratios showed significant increase with increasing fraction of cells in S-phase or significant decrease with increasing tumour volume-doubling time across the four xenograft lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effect of melanin on phosphorus T1S in human melanoma xenografts studied by 31P MRS.

31P MRS resonance ratios of tumors depend on the T1S of the phosphorus compounds. The objective of the 31P MRS study reported here was to investigate whether the phosphorus T1S of melanomas are influenced by the presence of melanin. One amelanotic (COX-t) and one melanotic (ROX-t) human melanoma xenograft line were studied at two different tumor volumes: 200 and 1000 mm3. 31P MRS was performed in nonanaesthetized mice at 4.7 T. The T1S were measured by using the superfast inversion recovery technique. Fraction of necrotic tissue in the tumors was determined by histological examination. The ROX-t tumors showed shorter T1S than the COX-t tumors at a volume of 200 mm3, where the fraction of necrotic tissue in the tumors was insignificant. The difference was similar in magnitude for all resonances. The T1S were not significantly different for COX-t and ROX-t at a volume of 1000 mm3, where the tumors of both lines had developed significant necrosis. The phosphorus T1S of melanomas without necrosis can be shortened significantly by the presence of melanin. The magnitude of the T1 shortening is similar for all major compounds. 31P MRS resonance ratios of melanomas are not altered significantly by correcting for effects of partial saturation.

Animals↗

PubMed1995

Lipases and esterases: a review of their sequences, structure and evolution.

This chapter aims to provide a brief review on the enzyme family of lipases and esterases. The sequences, 3D structures and pH dependent electrostatic signatures are presented and analyzed. Since the family comprises more than 100 sequences, we have tried to focus on the most interesting features from our perspective, which translates into finding similarities and differences between members of this family, in particular in and around the active sites, and to identify residues that are partially or totally conserved. Such residues we believe are either important for maintaining the structural scaf-fold of the protein or to maintain activity or specificity. The structure function relationship for these proteins is therefore of central interest. Can we uniquely identify a protein from this large family of sequences--and if so, what is the identifier? The protein family displays some highly complex features: many of the proteins are interfacially activated, i.e. they need to be in physical contact with the aggregated substrate. Access to the active site is blocked with either a loop fragment or an alpha-helical fragment in the absence of interfacial contact. Although the number of known, relevant protein 3D structures is growing steadily, we are nevertheless faced with a virtual explosion in the number of known or deduced amino acid sequences. It is therefore unrealistic to expect that all protein sequences within the foreseeable future will have their 3D structure determined by X-ray diffractional analysis or through other methods. When feasible the gene and/or the amino acid sequences will be analyzed from an evolutionary perspective. As the 3D folds are often remarkably similar, both among the triglyceride lipases as well as among the esterases, the functional diversities (e.g. specificity) must originate in differences in surface residue utilization, in particular of charged residues. The pH variations in the isopotential surfaces of some of the most interesting lipases are presented and a qualitative interpretation proposed. Finally we illustrate that NMR has potential for becoming an important tool in the study of lipases, esterases and their kinetics.

Amino Acid Sequence↗

PubMed1994

The blind watchmaker and rational protein engineering.

In the present review some scientific areas of key importance for protein engineering are discussed, such as problems involved in deducting protein sequence from DNA sequence (due to posttranscriptional editing, splicing and posttranslational modifications), modelling of protein structures by homology, NMR of large proteins (including probing the molecular surface with relaxation agents), simulation of protein structures by molecular dynamics and simulation of electrostatic effects in proteins (including pH-dependent effects). It is argued that all of these areas could be of key importance in most protein engineering projects, because they give access to increased and often unique information. In the last part of the review some potential areas for future applications of protein engineering approaches are discussed, such as non-conventional media, de novo design and nanotechnology.

Animals↗

PubMed1994

13C NMR spectroscopy study of cortical nerve cell cultures exposed to hypoxia.

Primary cultures of cerebral cortical GABA-ergic neurons growing on top of a preformed layer of astrocytes (co-cultures) were incubated with [1-13C]glucose and exposed to a low oxygen atmosphere (2% O2) for 17 hr. 13C, 1H, and 31P nuclear magnetic resonance (NMR) spectroscopy was performed on perchloric acid (PCA) extracts of cells and of media collected from these cultures. In the control groups incorporation of 13C label into glutamine, citrate, and lactate could be demonstrated in both cell extracts and culture media. Labeled GABA and glutamate were only observed in cell extracts. During hypoxia high energy phosphates decreased but lactate production and glucose consumption increased. There was a decreased amount of citrate and glutamine in cell extracts and media of the hypoxic co-cultures. There was a change in distribution of the 13C label within the GABA molecule, with an increase of labeling in the C-2 position. This change in 13C distribution was not found in glutamine present in the media where it is a precursor for GABA in neurons. Instead a decrease in the corresponding C-4 position was observed. These results suggest that energy depletion during hypoxia leads to reduced export from the astrocytic tricarboxylic acid (TCA) cycle as demonstrated by a decreased amount of citrate and changed distribution of 13C in glutamine. The change in the distribution of label in GABA from cell extracts as compared to glutamine in the medium may indicate that neurons are synthesizing GABA using precursors supplied from their own TCA cycle and not from precursors supplied by astrocytes.

Animals↗

PubMed1994

1H NMR spectroscopic characterization of perchloric acid extracts from breast carcinomas and non-involved breast tissue.

High-resolution 1H NMR spectra were obtained from perchloric acid extracts of breast tumor specimens and adjacent non-involved tissue. Two-dimensional shift-correlated and homonuclear J-resolved spectroscopy were used to identify coupled resonances in the spectra. Chemical shifts, multiplicities and spin-spin coupling constants of several non-resolved resonances in the one-dimensional spectra could be determined by the two-dimensional methods. Several differences in the metabolite content of the two types of extracts were established. The spectra of extracts from non-involved tissue were dominated by signals from glucose and other carbohydrates, while most of the tumors had very low or no detectable levels of glucose. High concentrations of lactate, taurine and succinate, an increase of the phosphocholine level, and a very low phosphocreatine level were characteristic findings in the 1H spectra of tumor extracts. The variation in the level of myo-inositol follows the variation in glucose for the two types of tissue. Scyllo-inositol was for the first time observed in the NMR spectra from breast tissue. Uridine 5'-diphospho-N-acetylglucosamine and uridine 5'-diphospho-N-acetylgalactosamine have been identified and there is an increased level of these two hexoses in the tumor tissue. These results provide insight into breast tumor metabolism, by simultaneously detecting a large number of metabolites and demonstrate the potential for using 1H NMR spectroscopy for studying different metabolic pathways in breast tumors. At the same time they provide useful information for interpretation of in vivo 1H NMR spectra of breast tumors.

Breast↗

PubMed1994

NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: compartmentation of astrocyte metabolism.

Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases). The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy. Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation. After 3 h of hypoxia full recovery was possible during reoxygenation. Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia. Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia. Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes. In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia. Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing [2-13C]acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors. The media were subsequently analyzed by 13C NMR spectroscopy. After shorter periods of hypoxia (3 h) full recovery was possible. Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia. 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged. These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia. Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios. During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.

Adenosine Triphosphate↗

PubMed1994

NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) did not affect recovery of high energy phosphates and pH in early reperfusion in a rat model of transient forebrain ischemia. Or: an in vivo 31P NMR spectroscopy study.

The new non-NMDA (N-methyl-D-aspartate) receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) has previously been shown to exert a neuroprotective effect in animal models of cerebral ischemia when administered in the post-ischemic phase. In this investigation the effect of NBQX on acidosis and energy recovery in early reperfusion after 10 min of transient forebrain ischemia with the 2-vessel occlusion model in the rat was studied with 31P NMR spectroscopy. In the intervention group the animals received a bolus dose of NBQX 30 mg.kg-1 i.v. at the start of reperfusion. 31P NMR spectroscopy was used to measure intracellular pH, ATP and phosphocreatine continuously in-vivo during, and after, the ischemic event. The recovery of high energy phosphates and pH was followed during 30 min of reperfusion. Pre-ischemic levels of phosphocreatine were reached after approximately 9-10 min in both groups. Although a slight improvement could be seen in the intervention group there was no significant difference in the rate of recovery between the two groups. ATP reached 90% of preischemic levels after about 8 min without significant difference between the two groups. With respect to the recovery of intracellular pH, no difference could be shown. Our results do not contradict previously published results, but suggest that the potential protective effect of NBQX is not mediated through improved recovery of energy metabolism in early reperfusion.

Acidosis↗

PubMed1994

Nuclear magnetic resonance spectroscopy: biochemical evaluation of brain function in vivo and in vitro.

Nuclear magnetic resonance spectroscopy (MRS) offers a unique opportunity to monitor mmolar concentrations of high energy phosphates, glucose, lactate and amino acids. The possibility of obtaining information about chemical constituents noninvasively is of great importance. MRS and chemical shift imaging (CSI) are emerging as tools for tumor grading, monitoring of treatment, ischemia research, in pediatric research for follow-up of children with borderline mental retardation, for defining brain death and to define epileptic foci. It is important to know which cell type (neuronal or glial) shows changes as a result of external manipulations (e.g. excitotoxins) or internal changes (brain pathology). Metabolic studies have been carried out on brain cell cultures. By using 13C labeled glucose and acetate in combination with 13C MRS it was shown that astrocytes release lactate, glutamine, citrate and alanine and that cerebral cortical neurons use glutamine released from astrocytes as a precursor for GABA synthesis. An important feature in MRS is the localization of N-acetyl aspartate in neurons, since this enables monitoring of neuronal reactions, such as survival after neurotoxic insults. Recent advances have yielded high speed functional echo planar imaging (EPI) techniques that are sensitive to changes in cerebral blood volume, blood flow and blood oxygenation (Functional MRI). During cognitive task performance, local alterations in neuronal activity induce local changes in cerebral metabolism and cerebral perfusion, which can now be detected with MRI.

Animals↗

PubMed1993

Protein structures from distance inequalities.

A computer method for folding protein backbones from distance inequalities is presented. It involves an algorithm that uses a novel approach for handling inequalities through the minimization of a continuous energy function. Tests of the folding algorithm have been carried out on a small protein, the 6PTI (bovine pancreatic trypsin inhibitor) with 56 amino acid residues, and on a medium-size protein, the 1TRM (rat trypsin) with 223 amino acid residues. Reconstructions based on a real-valued distance matrix led to folded three-dimensional structures with root-mean-square values of 0.04 A when compared with the crystallographic data. The obtained root-mean-square measures were of the order of 1 A, when distance inequalities were used for the reconstruction. Subsequently, the folding approach has been applied to distance inequalities predicted by neural network techniques that use the amino acid sequence as the only input. The inaccuracy in the inequalities predicted by the neural network was the reason for the root-mean-square value of 5.2 A. An error analysis of the method for reconstruction was performed and showed that no more than 3% inaccurate distance inequalities could be corrected for. Finally, a simple technique for root-mean-square comparisons of different protein structures is discussed.

Algorithms↗

PubMed1993

[AIDS--knowledge, behavior and attitude at the University of Aarhus in 1990. A questionnaire study].

To describe knowledge, attitude and behaviour regarding AIDS among students and employees at the University of Aarhus an anonymous self-administered questionnaire survey was conducted in April 1990. Of the 2169 and 850 questionnaires to students and employees, respectively, 1548 (71.4%) and 568 (67%) were returned. The study group had an excellent knowledge about AIDS and the transmission of HIV-virus. However, when defining "risk behaviour" in relation to transmission of HIV-virus among heterosexuals as "> or = 2 sexual partners within the past year without using condoms" 27% of the male and 20% of the female students showed risk behaviour. Among employees the rate was highest (23%) among the male scientific staff than among the remainder (17%). It is concluded that risk behaviour was not related to age and occurred in spite of the fact that 42% of the students and 31% of the employees, who showed risk behaviour were also aware of a personal risk of being infected. There is still a need for information about AIDS, and future campaigns should be directed towards all age groups as risk behaviour is not only a phenomenon among young people.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Direct demonstration by [13C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons.

Primary cultures of cerebral cortical astrocytes and neurons, as well as neurons growing on top of the astrocytes (sandwich co-cultures), were incubated with 1-[13C]glucose or 2-[13C]acetate and in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. [13C]NMR spectroscopy at 125 MHz was performed on perchloric acid extracts of the cells or on media collected from the cultures. In addition, the [13C/12C] ratios of the amino acids glutamine, glutamate and 4-aminobutyrate (GABA) were determined by gas chromatography/mass spectroscopy, showing a larger degree of labeling in GABA than in glutamate and glutamine from glucose. Glutamine and glutamate were predominantly labeled from acetate. A picture of cellular metabolism mainly regarding the tricarboxylic acid cycle and glycolysis was obtained. Due to the fact that acetate is not metabolized by neurons to any significant extent, it could be shown that precursors from astrocytes are incorporated into the GABA pool of neurons grown in co-culture with astrocytes. Spectra of media removed from these cultures revealed that likely precursor candidates for GABA were glutamine and citrate. The importance of glutamine is further substantiated by the finding that inhibition of glutamine synthetase, an enzyme present in astrocytes only, significantly decreased the labeling of GABA in co-cultures incubated with 2-[13C]acetate.

Acetates↗