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Biomedical subjects

G Sandberg

Publications and source records attributed to G Sandberg.

At least 55 records · Page 3Linked to original sources
PubMed1994

Reproducibility of three identification systems for biotyping of coagulase-negative staphylococci.

Three commercial identification systems were evaluated as tools for biotyping coagulase-negative staphylococci. Emphasis was placed on the reproducibility of component tests and not on the ability of these kits to identify these bacteria accurately. Forty-seven clinical and reference strains of Staphylococcus were tested in duplicate with each system. The Staph-Ident profile of test results changed for 20 strains on repeat testing, the Staph-Trac profile changed for 10 strains, and the Vitek GPI profile changed for 14 strains. The component tests of each system that were responsible for these profile changes were identified.

Bacterial Typing Techniques↗
PubMed1994

Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

5-Methylcytosine↗
PubMed1993

Effect of L-alanine and some other amino acids on thymocyte proliferation in vivo.

L-alanine was shown earlier to play a significant role for the proliferation of lymphocytes in vitro. In the present work the effect of L-alanine and some other amino acids on thymocyte proliferation was studied in vivo by local administration into one thymus lobe of guinea pigs. Proliferating cells were pulse labelled with bromodeoxyuridine (BrdUrd). The labelling index of the treated lobe significantly exceeded that of the contralateral, control lobe at 48 h after treatment with 10 or 100 micrograms L-alanine, indicating stimulated proliferation. The higher dose, which was also tested after other time intervals, stimulated also at 24 h. The difference in proliferative activity between the lobes was verified by mitotic studies. The effect of L-alanine was mainly on the large, low density, highly proliferating precursor cells. No other amino acids tested (D-alanine, cysteine, hydroxyproline, serine, tryptophan), or pyruvate produced significant differences between the treated and control lobes.

Alanine↗
PubMed1992

Analysis of Indole-3-Acetic Acid Metabolites from Dalbergia dolichopetala by High Performance Liquid Chromatography-Mass Spectrometry.

A mixture of [2-(14)C(1)] and [(13)C(6)]indole-3-acetic acid was applied to the cotyledons of 6-day-germinated seeds of "jacarandá do cerrado" (Dalbergia dolichopetala) and after 8 hours the seeds were extracted. Analysis of the fractionated extract by reversed-phase high performance liquid chromatography-radiocounting revealed the presence of five radiolabeled metabolite peaks (I-V). After further purification, the individual peaks of radioactivity were analyzed by combined high performance liquid chromatography-steel filter-fast atom bombardment-mass spectrometry. The metabolite fraction V was found to contain [(14)C(1), (13)C(6)]indole-3-acetylas-partic acid and unlabeled indole-3-acetylglutamic acid. Analysis of the metabolite fraction II revealed the presence of dioxindole-3-acetylaspartic acid and putative dioxindole-3-acetylglutamic acid as well as putative benzene ring-hydroxylated derivatives of oxindole-3-acetylaspartic acid and oxindole-3-acetylglutamic acid. There was no evidence of significant incorporation of label from [2'-(14)C(1)] or [(13)C(6)]indole-3-acetic acid into any of these conjugated indoles.

Journal Article↗
PubMed1992

Metabolism of Indole-3-Acetic Acid by Pericarp Discs from Immature and Mature Tomato (Lycopersicon esculentum Mill).

[1'-(14)C, (13)C(6)]Indole-3-acetic acid was infiltrated into immature pericarp discs from fruits of tomato (Lycopersicon esculentum Mill., cv Moneymaker). After a 24-h incubation period the discs were extracted with methanol and the partially purified extract was analyzed by reversed-phase high-performance liquid chromatography-radiocounting. Five metabolite peaks (1-5) were detected and subsequently analyzed by combined high-performance liquid chromatography-frit-fast atom bombardment-mass spectrometry. The metabolite 4 fraction was found to contain [(13)C(6)]-indole-3-acetylaspartic acid, and analysis of metabolite 5 identified [(13)C(6)]indole-3-acetyl-beta-d-glucose. The other metabolites could not be identified, but alkaline hydrolysis studies and gel permeation chromatography indicated that metabolites 1 and 3 were both amide conjugates with a molecular weight of approximately 600. Studies with radiolabeled indole-3-acetic acid, indole-3-acetylaspartic acid, and indole-3-acetyl-beta-d-glucose demonstrated that in immature pericarp indole-3-acetic acid is deactivated primarily via metabolism to indole-3-acetylaspartic acid, which is further converted to metabolites 1, 2, and 3. In mature, pink pericarp discs, indole-3-acetic acid is converted more extensively to its glucosyl conjugate. Conjugation of indole-3-acetic acid to indole-3-acetylaspartic acid appears to be dependent upon protein synthesis because it is inhibited by cycloheximide. In contrast, cycloheximide has little effect on the further conversion of indole-3-acetylaspartic acid to metabolites 1, 2, and 3.

Journal Article↗
PubMed1992

Transgenic Tobacco Plants Coexpressing the Agrobacterium tumefaciens iaaM and iaaH Genes Display Altered Growth and Indoleacetic Acid Metabolism.

Transgenic tobacco (Nicotiana tabacum) SR1 plants expressing the Agrobacterium tumefaciens nopaline transferred DNA iaaH gene were transformed with a 35S-iaaM construct. The transformants displayed several morphological aberrations, such as adventitious root formation on stem and leaves, dwarfism, epinastic leaf growth, increased apical dominance, and an overall retardation in development. In addition, xylem lignification was higher than in wild type. Free and conjugated indoleacetic acid (IAA) levels were quantified by gas chromatography-multiple ion monitoring-mass spectrometry in leaves and internodes of wild-type plants and two transformed lines with different phenotypes. Both transformed lines contained elevated levels of free and conjugated IAA, which was associated with increased transcription of the iaaM gene. The line with the highest IAA level also had the most altered pattern of growth and development. These IAA-overproducing plants will provide a model system for studies on IAA metabolism, IAA interactions with other phytohormones, and IAA roles in regulating plant growth and development.

Journal Article↗
PubMed1991

After microinjection hemimethylated DNA is converted into symmetrically methylated DNA before DNA replication.

In this investigation we analysed the maintenance methylation activity of the mammalian cell DNA methyltransferase by microinjection of hemimethylated HSV-tk DNA into thymidine kinase-negative rat 2 cells. We found that the hemimethylated DNA was efficiently converted into symmetrical methylated molecules before DNA replication. Furthermore, integration of the trans-DNA into the host genome is an early event after gene transfer.

Animals↗
PubMed1991

T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants.

Oncogenes carried by the transferred DNA (T-DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T-DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T-DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28-fold increase in conversion of tryptophan to indole-3-lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis-regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax-1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole-3-acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.

Base Sequence↗
PubMed1991

In vivo stimulation of thymocyte proliferation by thymocyte growth peptide (TGP).

Chromatographically pure thymocyte growth peptide (TGP) was injected into one of the two thymus lobes of guinea pigs. Pulse labelling of DNA-synthesizing cells was obtained by an intracardial injection of bromodeoxyuridine (BrdUrd). After 24 and 48 h, the frequency of labelled cells was increased in the TGP-treated lobe in comparison with the control lobe. At 24 h, the increase was found in non-rosetting, large, low-density cells, the population containing the highest frequency of DNA-synthesizing cells. At 48 h, labelled rosette-forming, small, high-density cells appeared in an increased frequency in the TGP-treated lobe. Serum thymic factor (FTS), which is structurally related to TGP, did not produce similar effects. The results show that TGP is active in vivo and stimulates proliferation of cycling thymic precursor cells which then are transformed into small thymocytes.

Animals↗
PubMed1991

Free and Conjugated Indoleacetic Acid (IAA) Contents in Transgenic Tobacco Plants Expressing the iaaM and iaaH IAA Biosynthesis Genes from Agrobacterium tumefaciens.

The Agrobacterium tumefaciens T-DNA gene iaaM was introduced by leaf-disc transformation into transgenic tobacco (Nicotiana tabacum) plants expressing the iaaH gene. Regenerated calli were screened for the presence of indole-3-acetamide (IAM), by gas chromatography-multiple ion monitoring-mass spectrometry, and IAM-containing calli were further analyzed for free and conjugated indoleacetic acid (IAA). It was found that transgenic calli on average contained twice as much free IAA and three times more conjugated IAA than calli from wild-type plants. About 40% of the transformed calli could be regenerated to plants. The distribution of free and conjugated IAA was measured in transformed plants with a normal phenotype and compared with equivalent wild-type plants. The IAA content of transgenic plants was only slightly increased, whereas IAA-conjugate levels were enhanced significantly. These data suggest that conjugation of IAA may serve as a regulatory mechanism, contributing to maintenance of steady-state IAA pool sizes during tobacco growth and development.

Journal Article↗
PubMed1991

Autogenic thymocytotoxic IgG antibody in normal guinea pigs.

Normal guinea pig serum (GPS) was confirmed to be cytotoxic for guinea pig thymocytes at 4 degrees C. Approximately 50% of the cells were sensitive for the cytotoxic action of GPS (4 h incubation). Binding of IgG and, to a smaller extent, IgM to the thymocyte surface was shown after incubation with GPS. Absorption of GPS with protein A agarose (which removes immunoglobulins), heat inactivation (56 degrees C, which removes complement activity) or treatment with 2-mercaptoethanol (2-me) (which affected binding of IgG and IgM to thymocytes) abolished the cytotoxic activity. After gel filtration chromatography with Sephadex G-150, the cytotoxic activity was found in fractions with molecular weights comparable to that of IgG. This is different from reports of others on natural thymocytotoxic antibodies (NTA) where IgM was found to be the dominant thymocytotoxic antibody. The toxic activity in GPS was readily absorbed by autogenic bone marrow, spleen, lymph node and thymus cells. In contrast to normal thymocytes, mitogen stimulated thymus cells were totally resistant to the cytotoxic activity. In summary, it is suggested that the thymocytotoxic activity in GPS at 4 degrees C is mediated by IgG and complement and is directed against immature thymocytes. The antigenic determinant is also present on cells in other lymphoid organs.

Animals↗
PubMed1990

Lymphatic vessels and Kurloff cells in the thymus of estradiol-treated guinea pigs.

Male guinea pigs were given a single subcutaneous injection of estradiol, which induces formation of Kurloff cells, and serial sections of thymus were examined after 10, 12, 15 and 21 days. Kurloff cells were found in large numbers in lymphatic vessels, both outside the thymus, in the interlobular tissue, at the cortical surface and inside the cortex, suggesting migration via such structures. Large extrathymic or interlobular lymphatics communicated with a previously undescribed thymic structure - the 'lymphatic centre' - surrounded by a marginal sinus. The orientation of lymphatic valves, and the concentration of Kurloff cells within this 'lymphatic centre' at an early time after the administration of estradiol, indicate the existence of an afferent migratory pathway. The different morphology at different times after estradiol suggest that the treatment caused a dynamic remodeling of thymic lymphatic structures.

Animals↗
PubMed1990

Abolished net output of lymphocytes from the spleen after immunization with Salmonella typhi H.

Cell release from the spleen was studied by comparing the content of mono- and polynuclear leucocytes in splenic afferent and efferent blood. A difference in the content of mononuclear cells was registered in normal animals, indicating a release of such cells from the spleen. Immunization with Salmonella typhi H antigen reduced this veno-arterial difference and abolished it totally after 11 days. Normal values were obtained again 28 days after immunization. The results indicate that the antigen causes an altered relation between input and output of mononuclear cells, mainly lymphocytes.

Animals↗
PubMed1989

Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi.

Luciferase (Lux)-encoding sequences are very useful as reporter genes. However, a drawback when applying Vibrio harveyi Lux as a reporter enzyme in eukaryotic cells, is that it is a heterodimeric enzyme, thus requiring simultaneous synthesis of both Lux subunits to be active. To overcome this disadvantage, luxA and luxB genes encoding the A and B subunits of this light-emitting heterodimeric Lux, were fused and expressed in Escherichia coli. Comparative analysis of four fused monomeric Lux enzymes by in vivo enzyme assay, immunoblotting and partial enzyme purification, showed that the fused Lux were active both as AB or as BA monomers, albeit at different levels (up to 80% activity for AB and up to 2% for BA, as compared with the wild type binary A + B construct). One of the LuxAB fusion proteins was stably expressed in calli of Nicotiana tabacum, and displayed very high Lux activity, thus demonstrating its potential as a reporter enzyme in eukaryotic systems.

Base Sequence↗
PubMed1989

Studies on thymocyte subpopulations in guinea pigs: in vivo differentiation of bromodeoxyuridine labelled cells, with special reference to rosette-forming ability.

Cycling thymocytes were labelled by an intracardial injection of bromodeoxyuridine (BrdUrd) in a total of 32 guinea pigs and the incorporation into DNA studied in subpopulations of cells defined by buoyant density and rosette-forming ability. The labelling pattern was compared at different times after injection (0.5 h to 120 h). A marked shift of labelled cells from large, low density cells (population 1a) to small, high density cells (population 2) was observed. During the first 48 h, the ratio between labelling indices of cell populations 1a and 2 decreased from 10 to 0.5. The number of labelled cells forming rosettes with rabbit erythrocytes (RFC+) increased while the number of labelled non-rosetting cells (RFC-) decreased from 0.5 h to 48 h, probably representing transformation of RFC- to RFC+. Then, after a decreased labelling in all cell populations at 72 h, an increase in both RFC+ and RFC- populations occurred at 96 h. The labelling in RFC- cells at 96 h was nearly as high as immediately after labelling. This second labelling of RFC- cells could represent immigration of precursor cells, a wave of proliferation in initially labelled precursors, and/or the formation of mature cells from the initially labelled precursors. The results indicate that a great majority of proliferating cells differentiate into small, high density cells within 48 h and that rosetting ability is acquired in many cells during this period. A model of intrathymic differentiation which fits the observations is presented.

Animals↗
PubMed1989

Effect of the neuropeptides beta-MSH, neurotensin, NPY, PHI, somatostatin and substance P on proliferation of lymphocytes in vitro.

Various neuropeptides were tested in a wide dose range for an effect on lymphocyte-proliferation. The response of spleen cells to the mitogen phytohaemagglutinin (PHA) was stimulated by somatostatin in doses ranging from 10(-6) to 10(-18) M. The effect of somatostatin was only seen when the peptide was added at the start of incubation, indicating an effect on the inductive phase of mitogen stimulation. Weak stimulative effects were also seen of somatostatin (10(-6) M) on spontaneous proliferation of thymocytes and of beta-MSH on PHA-treated thymocytes and PHA-treated spleen cells when the peptide was added after 24 h of culture.

Animals↗