Biomedical subjects

G Stenberg

Publications and source records attributed to G Stenberg.

31 records · Page 2Linked to original sources

PubMed1995

High-level bacterial expression of human glutathione transferase P1-1 encoded by semisynthetic DNA.

A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing the entire protein. The synthetic DNA sequence was constructed to achieve efficient base pairing with Escherichia coli 16S ribosomal RNA, avoidance of internal secondary structure, and optimal codon usage for high-level protein expression in accord with the known preferences in E. coli. The truncated GST P1-1 cDNA sequence and the synthetic segment were ligated into a plasmid to give an inducible expression system. Among the resulting clones a limited number was selected by immunodetection for highest yield of GST P1-1. Maximal expression was obtained from a spontaneously mutated sequence with altered as well as deleted bases as compared to the original construct. This clone, pKXHP1, allowed heterologous expression in E. coli in yields of > 200 mg enzyme per liter culture medium. The physicochemical and catalytic properties of the recombinant protein were indistinguishable from those of the enzyme purified from human placenta.

Base Sequence↗

PubMed1994

Ethanol reduces asymmetry of visual rCBF responses.

Visual regional CBF (rCBF) responses were measured in 10 healthy male subjects before and after an ethanol dose of 1 g/kg body weight. This dose induces well-established cerebral vasodilatation. However, significant bilateral occipital increases were found in both conditions. Apparently, the coupling between neuronal activity and rCBF is preserved following ethanol. The occipital and posterior parietal flow increases were, however, larger on the right than the left side in the sober state. During inebriation the asymmetry disappeared, possibly representing a more undifferentiated processing of visual information. We propose that ethanol causes a reduced inhibition of the left posterior cortex and a reduction of right-hemisphere information processing.

Adult↗

PubMed1994

EEG topography of acute ethanol effects in resting and activated normals.

Acute effects of ethanol on spectral characteristics of the EEG were studied using 18 recording sites and topographic mapping. The EEG was recorded both at rest and during a mental arithmetic task. Healthy young male volunteers were randomly assigned to an ethanol (n = 22) or a placebo (n = 15) group. The ethanol group received a total dose of 1.0 g/kg, divided into two equal doses given 75 minutes apart. and measurement sessions took place at baseline and after each dose. The placebo group underwent a similar schedule. Power in the theta, alpha and beta bands all increased in the ethanol group, but only the theta and beta bands clearly separated ethanol from placebo. Alpha increases were seen in the placebo group as well. The ethanol-induced changes were greater in the left hemisphere than in the right, having the effect of attenuating the right-over-left asymmetry seen at baseline. Differences between ethanol and placebo were more marked in the mentally activated condition, since the changes seen at rest were inhibited by the activation in the placebo group, but not in the ethanol group. The results indicate (1) that ethanol induces a less differentiated pattern of activity within the brain at rest, and (2) that it impairs the capacity to activate the brain under the challenge of a mental task.

Adult↗

PubMed1993

Motor imagery activates the cerebellum regionally. A SPECT rCBF study with 99mTc-HMPAO.

Our earlier findings of a cerebellar activation during motor imagery (Brain Res., 535 (1990) 313-317) were made with a technique with low regional resolution. Therefore we could not elucidate the distribution of the cerebellar activation. In the present study the cerebellar regional cerebral blood flow (rCBF) changes during motor imagery (MI) was measured with a single photon emission computed tomography (SPECT) rCBF method (99mTc-HMPAO) with higher regional resolution during (1) silent counting, and (2) MI (which included silent counting) in 17 normal subjects. Comparing the SPECT results from the two tasks revealed the regional activations during MI. We confirmed that the most pronounced regional activations during MI were found in the cerebellum, especially in its infero-lateral parts on both sides.

Adult↗

PubMed1993

Mapping of glutathione transferase (GST) genes in the rat.

Glutathione transferases (GST) make up a large group of related enzymes in mammalian tissues. The enzyme molecules are dimeric and at least 13 different subunits occur in the rat. Each subunit appears to be coded for by a distinct gene, and thus there is a large GST gene family in the rat. Recently, there have been several reports of the mapping of rat GST genes. In the present communication we confirm the previous assignments and extend the data with the mapping to rat chromosome 2 of a previously unmapped GST gene (Gstm1), and with the regional mapping of seven Gstp genes. These mappings provide further evidence for conservation of syntenic gene relationships among mammals. The human homologs of Gstm1 map to chromosome 1, and belong to a group of 9 genes that show conserved synteny on rat chromosome 2. The corresponding murine genes in most cases map to mouse chromosome 3. Similarly, the human homolog of Gstp maps to chromosome 11, and is one of 10 genes that exhibit conserved synteny on rat chromosome 1. The corresponding mouse genes map to mouse chromosome 7. Previously only one gene on rat chromosome 8 had a human homolog on chromosome 6, and rat Gsta1 is the second instance. Based on these mappings it appears that a new group of genes will exhibit conserved synteny on rat chromosome 8, human chromosome 6 and mouse chromosome 9. Interestingly, each of the three groups of conserved synteny seems to span the region across the centromeres of the human chromosomes.

Animals↗

PubMed1993

Acute effects of alcohol on regional cerebral blood flow in man.

Acute effects of alcohol in a low (0.7 g/kg) and a high dose (1.5 g/kg) on regional cerebral blood flow (rCBF) were measured with 133Xe inhalation technique at resting conditions in 13 normals. Mean hemisphere CBF increased globally by 12% at the lower dose and 16% at the higher dose. A normal hyperfrontal flow pattern was seen in both alcohol conditions. There were, however, significant regional differences in response to alcohol. The largest rCBF increase was observed in prefrontal regions at the lower dose, and in temporal regions at the higher. Expressed in relative values (% of the whole brain CBF), the temporal rCBF increased linearly with increasing alcohol dosage, while the prefrontal rCBF showed a increase at the lower dose followed by a decrease at the higher dose. It is concluded that alcohol has two types of acute effects on rCBF, a global vasodilatory effect and some regional effects, most clearly seen in prefrontal and temporal regions. The prefrontal flow augmentation following acute alcohol intake may be related to a transient arousal reaction, which has been reported by others. The temporal flow increase may be related to effects of alcohol on emotions and mood.

Adult↗

PubMed1992

Cloning and heterologous expression of cDNA encoding class alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic alpha,beta-unsaturated carbonyl compounds.

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha,beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

Amino Acid Sequence↗

PubMed1992

Heterologous expression of recombinant human glutathione transferase A1-1 from a hepatoma cell line.

A cDNA clone, lambda GTHA1, encoding human glutathione transferase A1-1 has been isolated from a hepatoma HepG2 cDNA library. At the nucleotide level, the new clone showed minor differences from cDNA deriving from normal liver, but the deduced amino acid sequence was identical to the structure previously described. The protein was expressed from a plasmid, pKHA1, and isolated by a single-step affinity purification on an S-hexylglutathione Sepharose matrix. The yield of the recombinant protein was 165 mg from a 3-liter culture of bacteria.

Amino Acid Sequence↗

PubMed1991

Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase.

Human class Alpha glutathione transferase (GST) A1-1 has been subjected to site-directed mutagenesis of a Tyr residue conserved in all classes of cytosolic GSTs. The change of Tyr8----Phe lowers the specific activities with three substrates to 2-8% of the values for the wild-type enzyme. The changes in the kinetic parameters kcat/KM, Vmax and S0.5 show that the decreased activities are partly due to a reduced affinity for glutathione. The effect is reflected in lowered kcat values, suggesting that the hydroxyl group of Tyr8 is involved in the activation of glutathione. The proposal of such a role for the Tyr residue has support from the 3D structure of a pig lung class Pi GST [Reinemer et al. (1991) EMBO J. 10, 1997-2005]. Thus, Tyr8 appears to be the first active site residue established as participating in the chemical mechanism of a GST.

Base Sequence↗

PubMed1991

Effects of directed mutagenesis on conserved arginine residues in a human Class Alpha glutathione transferase.

Glutathione transferase (GST) epsilon (also known as GST2 or GST B1B1), the major Class Alpha GST in human liver has been subjected to oligonucleotide-directed site-specific mutagenesis. Four arginine residues, R13, R20, R69 and R187, of which all but R69 are strictly conserved through GST Classes Alpha, Mu and Pi have been replaced by Ala. The mutant enzymes have been expressed in Escherichia coli, purified by affinity chromatography and characterised. Compared with the wild-type enzyme, all mutant GSTs had altered catalytic properties. All mutants had decreased specific activity with 1-chloro-2,4-dinitrobenzene (CDNB). Mutants R13A, R69A and R187A also showed decreased activities with other substrates such as cumene hydroperoxide (CuOOH) and androstenedione. In contrast, mutant R20A had an increased peroxidase activity and an isomerase activity essentially the same as that of the wild-type GST. With the substrates used, kcat./Km values were decreased for all mutant GSTs. Increases in the [S0.5] values were most significant for glutathione (GSH), while values for CDNB and CuOOH were less markedly affected. Thus, various kinetic data indicate that the GSH affinity has been reduced by the mutations and that this loss of affinity is linked to the decreased specific activities. Inhibition studies showed an increased sensitivity towards S-hexyl-GSH; this was particularly marked for mutant R69A. Mutant R20A had a lowered [I50] value but, in contrast, also the highest [I80] value as compared with the wild-type enzyme. Towards bromosulphophthalein, mutants R20A and R69A had a markedly increased sensitivity, about 35-fold in comparison with the wild-type. The inhibition properties of mutant R187A were similar to those of the wild-type enzyme and the properties of mutant R13A were in between. The increased sensitivity to S-hexyl-GSH, in contrast with the decreased affinity for GSH, was suggested to be due to an altered distribution between conformational states of the enzyme induced by the mutations. The arginine residues in positions 13, 20 and 69 all seem to be important for the catalytic properties of GST. Further, the inhibition studies indicate a role of arginine residues in the stabilisation of conformational states of the enzyme.

Amino Acid Sequence↗

PubMed1990

The cerebellum participates in mental activity: tomographic measurements of regional cerebral blood flow.

Measurements in man of regional cerebral blood flow (rCBF) have demonstrated a number of cortical and subcortical events coupled to sensory stimulation or motor performance. It has also been shown that local activity changes take place in the cortex during 'pure' mental activity such as motor imagery (unaccompanied by sensory input or motor output). Thus, our group has previously shown that imagination of hand movements gives predominantly a frontal cortical rCBF activation while the corresponding hand movement activates the rolandic hand area mainly. In this paper we report tomographic rCBF measurements with a 133-Xenon SPECT technique during imagined tennis movements and silent counting. Both procedures gave rise to a significant cerebellar activation in addition to cortical rCBF changes. Apparently, the cerebellum may participate in pure mental activity. It possibly plays a role for the temporal organization of neuronal events related to cognition.

Adult↗

PubMed1990

Neurophysiological changes during insulin-induced hypoglycaemia and in the recovery period following glucose infusion in type 1 (insulin-dependent) diabetes mellitus and in normal man.

Hypoglycaemia (median venous blood glucose 1.8 mmol/l; range 1.6-2.3) was induced by an intravenous infusion of regular insulin in eight patients with Type 1 (insulin-dependent) diabetes mellitus (age 28.0 +/- 7.4 years; mean +/- SD, duration 15.5 +/- 5.1 years) and in 12 age-matched healthy male control subjects. Multi-channel frequency analysis of electroencephalogram (electrophysiologic brain mapping) and recording of P300 and somatosensory evoked potentials were performed before, during and immediately after the hypoglycaemic period. The hypoglycaemia produced a significant increase in low frequency electroencephalographic activity in both groups, most pronounced over anterior regions of the brain. The electroencephalographic activity was normalised immediately after the hypoglycaemic period. The patients with diabetes showed somewhat longer P300 latencies during the initial normoglycaemic examination. Hypoglycaemia caused a marked reduction of the P300 amplitude in both groups of subjects and the amplitude was not restored immediately after normalisation of blood glucose levels. The somatosensory cortical responses were not affected by hypoglycaemia. We conclude that hypoglycaemia results in impairment in cerebral function, as measured by neurophysiological techniques, which is not immediately normalised when blood glucose is restored to normal.

Adult↗

PubMedDate not supplied

Quantified EEG and cortical evoked responses in patients with chronic traumatic frontal lesions.

Eighteen frontal trauma patients and 17 age-matched control subjects had quantified EEGs and measurements of sensory (SEP) and auditory evoked potentials (P300) using a Biologic Brain Atlas III system. The findings were compared to the conventional paper EEG, and to the frontal lesion volumes, severity of head injury, and outcome variables. The quantified EEG confirmed the pathological findings detected by visual inspection, but some regional abnormalities were more easily detected by topographic mapping. The regional distribution of pathological slowing corresponded well with the morphological lesions in most patients. The modal frequency of EEG correlated both with lesion volume and injury severity and with the outcome variables. There were no pathological findings in the SEPs, and all but one patient had clearly distinguishable P300 responses. There was a significant reduction in P300 amplitude in the frontal patients at the anterior, but not at the posterior electrodes. The topographical distribution of the P300 changes corresponded well with the morphological lesions. Our findings indicate that the P300 potential is, in part, dependent upon the prefrontal cortical areas. The present study thus supports P300 investigations which have shown amplitude reduction in other disorders (e.g., schizophrenia) with a presumed prefrontal dysfunction.

Adult↗