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Biomedical subjects

A Hamann

Publications and source records attributed to A Hamann.

At least 37 records · Page 2Linked to original sources
PubMed2003

Therapeutic dose of HIV-1 protease inhibitor saquinavir does not permanently influence early insulin signaling.

UNLABELLED: The introduction of HIV-1 protease inhibitor therapy has significantly improved the expectancy and quality of life for HIV-infected patients. Recent reports have highlighted the development of metabolic complications in patients taking protease inhibitors, including abnormalities in glucose metabolism such as impaired glucose tolerance and type 2 diabetes. The mechanisms by which protease inhibitors induce these metabolic syndromes are not well understood. The aim of this study was to determine whether treatment with the HIV-1 protease inhibitor, saquinavir, influences the early insulin signaling cascade in insulin-sensitive cell lines. METHODS: Insulin-stimulated phosphorylation of insulin receptor (IR-beta), insulin receptor substrates (IRS-1 and IRS-2), association of phosphatidylinositol 3-kinase (PI 3-kinase), Ser 473-phosphorylation of Akt and Thr202/Tyr204-phosphorylated p44/42 MAP kinase in 3T3L1 adipocytes and FAO hepatoma cells incubated with increasing concentrations of saquinavir for 24, 36 hours, 2, 3 and 6 days were measured. Results. Phosphorylation of IR-beta, IRS-1 and IRS-2 was not permanently affected by incubation with therapeutic doses (2.5 microM) of saquinavir for 36 hours. After 24 hours we observed an increase of IR-beta and IRS-1 phosphorylation. However, this initial stimulation of IR-beta and IRS-1 phosphorylation was not permanent and did not result in an increased PI 3-kinase association. Phosphorylation of IRS-2 and MAP kinase as well as glucose transport activity was not altered by therapeutic doses. Doses of 10, 25 and 50 microM of saquinavir altered the early insulin signaling events in a dose-dependent manner. However, this effect was primarily due to the cytotoxic effect of higher saquinavir doses. Glucose transport activity was not significantly reduced in 3T3L1 cells treated with 2.5 microM saquinavir in comparison to the control cells stimulated with insulin. CONCLUSION: Early insulin signaling cascade, essential for normal glucose metabolism, is not affected by therapeutic doses of saquinavir. The reduction of insulin-induced phosphorylation in higher concentrations is primarily related to cytotoxic effects. Other mechanisms than early insulin signaling must be primarily responsible for the metabolic alterations during saquinavir therapy.

3T3 Cells↗
PubMed2002

Microflora reactive IL-10 producing regulatory T cells are present in the colon of IL-2 deficient mice but lack efficacious inhibition of IFN-gamma and TNF-alpha production.

BACKGROUND: Inflammatory bowel disease in interleukin 2 (IL-2) deficient (IL-2(-/-)) mice is triggered by the intestinal microflora and mediated by CD4(+) T cells. AIMS: To determine the characteristics of microflora specific intestinal T cells, including migration and cytokine production. METHODS: Intestinal T cell populations and cytokine mRNA expression of specific pathogen free (SPF) and germ free (GF) IL-2(-/-) and IL-2(+/+) mice were compared by flow cytometry and reverse transcription-polymerase chain reaction. Cytokine production of intestinal mononuclear cells on stimulation with microflora antigens was assessed by ELISA. In vivo migration of T cells was assessed by adoptive transfer of (51)Cr labelled CD4(+)CD25(-)alpha beta(+) T cells. The ability of intestinal T cell lines to promote colitis was determined by adoptive transfer experiments. RESULTS: SPF IL-2(-/-) mice produced higher interferon gamma (IFN-gamma) and tumour necrosis factor alpha mRNA levels than GF IL-2(-/-) mice, which was accompanied by an increased number of CD4(+)alpha beta T cells in the colon. Tracking of (51)Cr labelled and adoptively transferred T cells revealed an increased MAdCAM-1 dependent but VCAM-1 independent recruitment of these cells into the colon of SPF IL-2(-/-) mice. Colon lamina propria lymphocytes (LPL) from SPF IL-2(-/-) mice showed increased spontaneous IFN-gamma production in vitro. On stimulation with bacterial microflora antigens, intraepithelial lymphocytes and LPL did not produce IFN-gamma, but high quantities of IL-10, which did not suppress IFN-gamma production. Bacterial antigen specific cell lines established from colon LPL of SPF IL-2(-/-) mice with colitis showed a regulatory T cell-like cytokine profile and only marginally modulated the course of colitis and survival of IL-2(-/-) mice. CONCLUSIONS: Our results suggest that microflora reactive regulatory T cells are present in the colon of SPF IL-2(-/-) mice. However, IL-10 produced by these cells did not significantly modulate a possible secondary proinflammatory CD4 Th1 cell population to produce IFN-gamma.

Adoptive Transfer↗
PubMed2001

[Long-term weight reduction. Current therapy approaches in obesity].

A large percentage of obese patients can lose weight with a combination of various dietary measures. The following possibilities are available: 1. The fat-reduced, carbohydrate-enhanced diet. 2. Formula diets, which are applied when a mixed diet has failed, or, alternatively, as an "introduction" to a weight-reducing program. In addition, suitable exercise programs and behavior-modifying measures aimed at stabilizing weight losses over the long-term, are also recommended. If this basic program fails, adjunctive drug treatment is indicated. Surgical treatment may be considered when various conservative forms of treatment have proved unsuccessful over a period of years.

Behavior Therapy↗
PubMed2001

[Molecular principles of obesity].

BACKGROUND: The deposition of excess amounts of energy in adipose tissue is enhanced by high-fat diets and lack of physical activity. Furthermore, the existence of a specific genetic predisposition towards the development of obesity becomes evident by marked interindividual differences in the response to caloric oversupply. GENETIC DEFECTS AND HORMONES: In recent years, numerous genes and genetic defects with importance for human obesity were identified, especially through studies in animal models. The adipocyte-derived hormone leptin and its hypothalamic receptor play a premier role, as they interact with a network of proteins and neuropeptides within the regulation of food intake and energy expenditure. CONCLUSION: The search for the key molecular mechanisms in the pathogenesis of obesity will not only improve our understanding of energy metabolism, but may ultimately also lead to the development of new treatment strategies for obese patients.

Adipose Tissue↗
PubMed2001

Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina.

The lifespan of the ascomycete Podospora anserina was previously demonstrated to be significantly increased in a copper-uptake mutant, suggesting that copper is a potential stressor involved in degenerative processes. In order to determine whether changes in copper stress occur in the cells during normal aging of cultures, we cloned and characterized a gene coding for a component of the molecular machinery involved in the control of copper homeostasis. This gene, PaMt1, is a single-copy gene that encodes a metallothionein of 26 amino acids. The coding sequence of PaMt1 is interrupted by a single intron. The deduced amino acid sequence shows a high degree of sequence identity to metallothioneins of the filamentous ascomycete Neurospora crassa and the basidiomycete Agaricus bisporus, and to the N-terminal portion of mammalian metallothioneins. Levels of PaMt1 transcript increase in response to elevated amounts of copper in the growth medium and during aging of wild-type cultures. In contrast, in the long-lived mutant grisea, transcript levels first increase but then decrease again. The ability of wild-type cultures to respond to exogenous copper stress via the induction of PaMt1 transcription is not affected as they grow older.

Agaricus↗
PubMed2001

Identification of a deletion variant in the gene encoding the human alpha(2A)-adrenergic receptor.

OBJECTIVE: The alpha(2)-adrenergic receptors are involved in the effects of catecholamines on energy metabolism. Of three known subtypes with differential expression, alpha(2A)-adrenergic receptors are also localized in adipose tissue where they counteract the lipolytic activity of beta-adrenergic receptors. This study was undertaken to assess whether variants in the alpha(2A)-adrenergic receptor gene are associated with body weight. DESIGN AND METHODS: Single strand conformation polymorphism (SSCP) screening and subsequent sequencing were applied to determine genetic variants in DNA samples from individuals with obesity, those of normal weight and those underweight. RESULTS: Analysis of the coding region resulted in the identification of an 18 bp deletion, with no other mutation found. Of 429 genotyped subjects, 7 carried the deletion, with no significant differences between lean and obese subjects. A previously identified polymorphism in the promoter of the alpha(2A)-adrenergic receptor gene also did not show an association with any of the tested body weight categories. CONCLUSION: Our data suggest that variants in the alpha(2A)-adrenergic receptor gene are unlikely to contribute to the predisposition for the lean or obese state.

Adolescent↗
PubMed2000

Transfer of IFNgamma-depleted CD4(+) T cells together with CD8(+) T cells leads to rejection of murine kidney sarcoma in mice.

In the murine kidney sarcoma, vaccination with the tumor-specific large T antigen induces protective immunity against the tumor. Immunity is dependent both on CD8(+) cytotoxic T cells and on CD4(+) T-helper cells. We analyzed whether the cytokine phenotype of induced CD4(+) T-effector cells might determine whether or not the tumor is successfully rejected. By intracytoplasmic staining of CD4(+) cells, IFNgamma-producing (Th1), IL-4-producing (Th2), and IL-10-expressing cells could be identified in vaccinated and non-vaccinated animals responding to tumor growth. Vaccinated mice rejecting the tumor showed an increase in the percentage of IL-4-producing (Th2) cells. In contrast, in non-vaccinated mice succumbing to the tumor, the immunosuppressive IL-10-producing cells became more abundant and the frequency of IFNgamma-expressing cells dropped at later time points. Yet, dominance by either a Th1 or a Th2 response could not be observed. To further clarify the relevance of these subsets, Th1 cells were enriched by cell sorting according to IFNgamma surface expression. Enriched Th1 and depleted cells, mainly consisting of the Th2 phenotype, were transferred together with CD8(+) T cells. Surprisingly, immunity could be transferred either with Th1 or Th2 cells, but Th2 cells were slightly more efficient. This suggests that, at least in the effector phase, a Th1 phenotype is not crucial for the rejection. Our findings support the view that the Th1/Th2 dichotomy is not central in T-cell-mediated tumor rejection.

Animals↗
PubMed2000

Activation induces rapid and profound alterations in the trafficking of T cells.

Activation and differentiation of lymphocytes have profound effects on their trafficking. Whereas naive T cells recirculate through lymphoid organs, activated cells localize predominantly in other compartments. Here, we report that changes in migratory properties of T cells occur immediately upon activation via the TCR. One hour stimulation is enough to target T cells into lung and liver following i.v. injection. The high localization within lung and liver and the lack of recirculation through lymphoid tissues are key features of activated lymphocytes. the source, in vitro as well as in vivo activated lymphocytes show this behavior, which is not caused by increased cell size. Accumulation in the lung requires protein synthesis and is partly mediated by LFA-1, in contrast to the acquisition of liver "homing" properties. Intravital microscopy reveals firm adhesion of activated cells within periportal sinusoids of the liver. Selective homing to other organs, such as skin or mucosa, was not observed, regardless of the cell's origin. These data indicate that activation quickly switches the trafficking program of lymphocytes from recirculation to sequestration; it is tempting to speculate that especially the induced trapping in the liver has a distinct role in limiting systemic T cell responses.

Animals↗
PubMed2000

Yeti--a degenerate gypsy-like LTR retrotransposon in the filamentous ascomycete Podospora anserina.

In the filamentous ascomycete Podospora anserina a 6,935-bp retrotransposon, Yeti, has been identified and characterized. It is flanked by a 5-bp target site duplication and contains long terminal repeats (LTRs) 354 bp in length. The LTRs show a high degree of identity to the previously reported repetitive element repa, a sequence suggested to represent a solo-LTR element of an unknown transposon. In the investigated Podospora strains, the number of complete Yeti copies is significantly lower than the number of repa elements, with up to 25 copies. Yeti appears to be inactive: it is highly degenerate and no transcripts of the element have been detected even in Podospora cultures grown under elevated stress conditions. The amino acid sequences deduced from Yeti display significant homology, particularly in the reverse transcriptase region, to those of other fungal retrotransposons, indicating that it is a member of the gypsy family. As suggested by the unusual dinucleotide content, degeneration of Yeti appears to be the result of a molecular mechanism resembling repeat-induced point mutation in Neurospora crassa.

Amino Acid Sequence↗
PubMed2000

The degenerate DNA transposon Pat and repeat-induced point mutation (RIP) in Podospora anserina.

A degenerate DNA transposon, Pat, was identified in the genomes of various wild-type strains of the filamentous fungus Podospora anserina. In these strains, the number (approximately 20-25 copies per genome) and location of Pat sequences appear to be conserved. Two copies of Pat, one complete and one partial, were cloned and characterized. The sequence of the complete element is 1856 bp long and contains imperfect inverted terminal repeats (ITRs) of 53 bp. The target site duplication comprises the sequence TA. The amino acid sequence derived from one reading frame of Pat shows significant homology to members of the Fot1 family of transposons. However, this reading frame is interrupted by numerous stop codons. Since no transcripts of Pat were identified in different P. anserina strains grown under standard conditions and under increased stress, we conclude that none of the copies of Pat is active in the strains analyzed, under the environmental conditions investigated. Comparison of the sequences of the two cloned Pat sequences revealed 89% (589/747 nucleotides) identity. Most of the differences (82%, 129/158) can be attributed to transitions preferentially at CpA:TpG and CpT:ApG dinucleotides. The dinucleotide ratios in Pat are similar to those in a Neurospora crassa transposon which was subject to repeat-induced mutation (RIP), but differ significantly from those found in single-copy genes of P. anserina and in fungal DNA transposons not modified by this mechanism. Molecular analysis of the progeny of a cross between the wild-type strain and a transgenic strain in which a nuclear gene was duplicated by transformation yielded the first clear evidence that a RIP-like process is active in P. anserina.

Amino Acid Sequence↗
PubMed2000

[Etiology of overweight and obesity].

Obesity results from a predominance of caloric intake over energy expenditure. Twin, adoption and family studies have demonstrated that, together with environmental conditions, various genetic factors play an important role in the pathogenesis of obesity. In recent years, it was possible to identify several defects in single genes responsible for obesity in rodents, some of which may also be involved in human obesity. Besides leptin as the most notable example, numerous other proteins and neuropeptides have been identified in recent years that participate in a complex network to regulate food intake and energy expenditure. The ongoing search for the important obesity genes should not only result in a better understanding of energy metabolism, but may also help in the development of new strategies for the treatment of obese patients.

Appetite Regulation↗
PubMed2000

Association of impaired phosphatidylinositol 3-kinase activity in GLUT1-containing vesicles with malinsertion of glucose transporters into the plasma membrane of fibroblasts from a patient with severe insulin resistance and clinical features of Werner syndrome.

The purpose of this study was to examine the molecular mechanism responsible for the defective insulin-stimulated glucose transport in cultured fibroblasts from a patient (VH) with clinical features of Werner syndrome and severe insulin resistance. Thus, in cells derived from VH, the subcellular distribution, structure, functional activity, as well as plasma membrane insertion of GLUT1 glucose transporters were analyzed. Furthermore, the insulin signal transduction pathway leading to activation of phosphatidylinositol (PI) 3-kinase as well as components of GLUT1-containing membrane vesicles were characterized. In fibroblasts derived from VH, GLUT1 glucose transporters were overexpressed by 8-fold in plasma membranes (PM) and by 5-fold in high density microsomes, respectively. Exofacial photolabeling revealed that only 14% of the overexpressed PM-GLUT1 transporters were properly inserted into the plasma membrane. The complementary DNA structure of the patient's insulin receptor and the GLUT1 glucose transporter, the intrinsic activity of plasma membrane glucose transporters, the tyrosine phosphorylation, as well as the protein expression of insulin receptor substrate-1/2 and p85 alpha/beta- and p110 alpha/beta-subunits of PI 3-kinase were normal. However, insulin-stimulated association of the p85 subunit of PI 3-kinase was defective in fibroblasts derived from VH compared to those from controls, and this defect was associated with a reduced IRS-1-dependent activation of PI 3-kinase by 50.2% and 63.6% after incubation for 5 and 10 min with 100 nmol/L insulin, respectively. Furthermore, immunodetection of small GTP-binding Rab proteins in subcellular membrane fractions indicated a decreased expression of Rab4 in total cellular homogenates as well as in high density microsomes by 70% and 58%, respectively. After preparation of GLUT1-containing vesicles, Rab4 was not detected to be a component of these vesicles. Analysis of the PI 3-kinase in GLUT1-containing membrane vesicles revealed insulin-dependent targeting of the p85 subunit to the vesicles immunoadsorbed from VH and control fibroblasts. Importantly, the association of the p85 subunit as well as the p85-immunoprecipitable PI 3-kinase activity were markedly reduced in GLUT1-vesicles derived from the patient. In conclusion, impaired PI 3-kinase activity in GLUT1-containing membrane vesicles derived from fibroblasts of VH is associated with a defective docking and/or fusion process of glucose transporters with the plasma membrane and thus might contribute to the molecular defect causing insulin resistance in this patient.

Adult↗
PubMed1999

Lymphocyte migration in lymphocyte function-associated antigen (LFA)-1-deficient mice.

Using lymphocyte function-associated antigen (LFA)-1(-/-) mice, we have examined the role of LFA-1 and other integrins in the recirculation of lymphocytes. LFA-1 has a key role in migration to peripheral lymph nodes (pLNs), and influences migration into other LNs. Second, the alpha4 integrins, alpha4beta7 and alpha4beta1, have a hitherto unrecognized ability to compensate for the lack of LFA-1 in migration to pLNs. These findings are confirmed using normal mice and blocking LFA-1 and alpha4 monoclonal antibodies. Unexpectedly, vascular cell adhesion molecule (VCAM)-1, which is essential in inflammatory responses, serves as the ligand for the alpha4 integrins on pLN high endothelial venules. VCAM-1 also participates in trafficking into mesenteric LNs and Peyer's patch nodes where mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the alpha4beta7-specific ligand, dominates. Both alpha4beta1, interacting with ligand VCAM-1, and also LFA-1 participate in substantial lymphocyte recirculation through bone marrow. These observations suggest that organ-specific adhesion receptor usage in mature lymphocyte recirculation is not as rigidly adhered to as previously considered, and that the same basic sets of adhesion receptors are used in both lymphocyte homing and inflammatory responses.

Animals↗