PubMed Health⌕ Search

Biomedical subjects

H Ostergaard

Publications and source records attributed to H Ostergaard.

At least 19 recordsLinked to original sources
PubMed2001

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Crystallography, X-Ray↗
PubMed2000

Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins. Cloning and characterization of five major molecular forms.

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers. WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins. WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) m(-1) s(-1)). None of the five serpins inhibited Bacillus subtilisin A, Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

Amino Acid Sequence↗
PubMed1996

A physical interaction between the cell death protein Fas and the tyrosine kinase p59fynT.

The Fas antigen (Apo1/CD95) is a transmembrane protein belonging to the nerve growth factor receptor family. It is expressed on a variety of cells, including activated T lymphocytes. Ligation of Fas with its natural ligand or with anti-Fas antibodies often results in the apoptotic death of the cell, making Fas an important mediator of down-regulating immune responses. The signal transduction pathways utilized by Fas are currently unknown, although tyrosine kinase activity has recently been strongly implicated. Here, we report that the tyrosine kinase p59fyn physically associates with Fas in Fas-sensitive cells. In addition, we show that activated T lymphocytes from fyn knockout mice exhibit elevated lifespans and reduced apoptosis in vitro compared to their normal counterparts. Furthermore, activated T lymphocytes from the fyn-deficient mice are less sensitive to killing by both anti-Fas antibody and Fas-ligand cytotoxic T cells. These results suggest that p59fyn plays an important role in Fas signal transduction.

Amino Acid Sequence↗
PubMed1995

Myxoma virus and Shope fibroma virus encode dual-specificity tyrosine/serine phosphatases which are essential for virus viability.

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SFV) led to the discovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were designated I1L and M1L, respectively. The localization and orientation of the myxoma I1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the Orthopoxviruses vaccinia virus and variola virus. The myxoma I1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro. To assess whether this is a common feature of poxvirus phosphatases, myxoma I1L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma I1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type I1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma I1L phosphatase, we constructed a recombinant myxoma virus containing a disrupted I1L open reading frame. This I1L mutant virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma I1L dual specificity phosphatase is an essential factor for virus viability.

Amino Acid Sequence↗
PubMed1995

Clinical applications of growth hormone for ovarian stimulation.

The realization of the existence of an intra-ovarian regulating mechanism involving insulin-like growth factor-I (IGF-I), a mediator of growth hormone (GH) action that augments the ovarian response to gonadotrophins, has prompted a number of clinical trials exploring the use of GH as an adjuvant for ovarian stimulation with human menopausal gonadotrophin. A critical review of these studies pinpoints a select group of infertile patients who may benefit from this co-treatment, particularly those who have a surgical, pathological or medically induced dysfunction of GH kinetics. The mechanism of this action, the effective dose needed and the implications regarding the interface of GH, IGF-I and ovarian physiology and pathology are now becoming clearer. A greater understanding of GH action on the ovary may in future benefit patients afflicted by anovulatory infertility and those requiring ovulation induction for in-vitro fertilization.

Clinical Trials as Topic↗
PubMed1992

The role of treatment with growth hormone in infertile patients.

The reality of the interaction of GH and its mediator, IGF-I, with gonadotrophins is now established. The results of these studies and others have obvious implications, both physiologically and clinically. Co-treatment with GH augments the gonadal response to gonadotrophins, and it seems to be of particular value in patients who are poor responders to gonadotrophin treatment and who have pituitary hypofunction induced surgically, idiopathically (hypogonadotrophic hypogonadism) or medically (treatment with GnRH analogues). There is conflicting evidence as to whether the observed effect of GH is exerted directly on the ovary or mediated through IGF-I. Treatment with GH causes a distinct increase in serum IGF-I concentrations, which correlate with, but are always higher than, follicular fluid levels, suggesting that GH stimulates hepatic production of IGF-I and that the effect on the ovary is endocrine. Further research will, hopefully, clearly define the precise therapeutic role of GH in the induction of ovulation, the selection of the most appropriate group of patients to be treated, and the minimum dose of GH needed to sensitize the ovary. Further studies are also needed to explore the action of GH and to define the role of IGF-I in the process of follicular development.

Adult↗
PubMed1992

Cotreatment with growth hormone for induction of spermatogenesis in patients with hypogonadotropic hypogonadism.

OBJECTIVE: To induce spermatogenesis by cotreatment with growth hormone (GH) and gonadotropin therapy in patients with hypogonadotropic hypogonadism who had failed to respond adequately to conventional treatment. DESIGN: Cotreatment with GH (4 IU) and human menopausal gonadotropin, 150 IU of follicle-stimulating hormone and 150 IU of luteinizing hormone (LH), three times a week, and human chorionic gonadotropin, 2,500 IU, two times a week for 24 weeks after unsuccessful treatment for 12 weeks with either pulsatile LH-releasing hormone or gonadotropins. SETTING: Specialist Reproductive Endocrine Unit. PATIENTS, PARTICIPANTS: Seven patients, four of whom had failed to respond adequately to the conventional treatment. MAIN OUTCOME MEASURES: Serum testosterone (T), estradiol, and sperm production, testicular and semen volume, and serum insulin-like growth factor-I and inhibin concentrations. RESULTS: Of the four patients who received cotreatment with GH, three increased T secretion (greater than 11 nmol/L) within a relatively short period of time, two produced adequate amount of sperm (13 and 12 x 10(6)/mL), and one of them impregnated his wife. One patient did not respond. CONCLUSION: The results offer a new approach to the problem of induction of spermatogenesis in patients who respond poorly to conventional treatment because cotreatment with GH enhanced T secretion and sperm production in a relatively short period of time.

Adult↗
PubMed1991

Cotreatment with growth hormone, after pituitary suppression, for ovarian stimulation in in vitro fertilization: a randomized, double-blind, placebo-control trial.

OBJECTIVE: To explore the effect of cotreatment with growth hormone (GH) for ovarian stimulation after pituitary suppression. DESIGN: A randomized, double-blind, placebo-controlled study. SETTING: Specialist Reproductive Endocrine and In Vitro Fertilization (IVF) Unit. PATIENTS, PARTICIPANTS: Twenty-five IVF patients who had responded suboptimally in a previous treatment cycle. A subgroup of 18 patients were found to have ultrasound (US) findings of polycystic ovaries (PCO). MAIN OUTCOME MEASURE: The amount of gonadotropin used, development of follicles greater than or equal to 14 mm, number of oocytes collected, fertilized, cleaved and replaced, serum and follicular fluid (FF) insulin-like growth factor I (IGF-I) concentrations. RESULTS: Cotreatment with GH was associated with a significant reduction in gonadotropins requirement (P less than 0.05). In patients with US-diagnosed PCO more follicles developed (P less than 0.05), more oocytes were collected (P less than 0.03), fertilized (P less than 0.004), and cleaved (P less than 0.02). A significantly higher FF IGF-I concentrations were found in patients receiving cotreatment with GH compared with those who received placebo (P less than 0.04). CONCLUSION: We believe that there may be a place for GH treatment in selected IVF cycles after pituitary suppression but what the role of IGF-I should further be investigated.

Adult↗
PubMed1991

Combined growth hormone and gonadotropin treatment for ovulation induction in patients with non-responsive ovaries.

Four anovulatory patients who did not respond to large doses of hMG over 18-33 days were co-treated in subsequent cycles with human growth hormone. Treatment with growth hormone markedly raised serum insulin-like growth factor concentrations. Two patients had a dramatic ovarian response within 7 days of co-treatment; two menopausal patients did not respond. This combined therapy may be of practical value for ovulation induction in non-menopausal patients with non-responsive ovaries.

Addison Disease↗
PubMed1991

Regulation of CD45 expression in human leukemia cells.

The CD45 antigen cluster identifies a family of transmembrane glycoprotein tyrosine phosphatases (PTPases) present on nearly all hemopoietic cells. Recent studies suggest that CD45 may play a role in the control of receptor mediated blood cell responses, and that expression of the CD45 gene varies during bone marrow cell maturation. However, relatively little is known of the mechanisms controlling CD45 expression and function. Here we show that the induction of granulocyte or monocyte differentiation of HL60 leukemia cells is accompanied by a rapid increase in CD45 antigen expression and CD45 PTPase activity. In contrast, other leukemia cell lines induced for monocyte/macrophage differentiation did not show increased CD45. Immunoprecipitation of radiolabelled CD45 glycoprotein from dimethyl sulphoxide (DMSO) treated HL60 cells indicated that the cells expressed 200 and 180 kD isoforms. Northern blots of steady-state RNA from HL60 cells showed a 4-11-fold increase in CD45 transcripts after DMSO treatment, but no alteration in the half-life of CD45 mRNA. Nuclear transcription assays showed that CD45 expression was controlled at the level of gene transcription. Namalwa Burkitt leukemia cells expressing the heterologous epidermal growth factor (EGF) receptor protein tyrosine kinase were used to assess the specificity of CD45 PTPase activity. Co-clustering of CD45 and the EGF receptor with specific monoclonal antibodies failed to alter the EGF stimulated tyrosine phosphorylation of the EGF receptor. These studies indicate that CD45 increases during myeloid maturation, and the expression of the CD45 gene is controlled at the level of gene transcription. Preliminary studies suggest that CD45 does not alter the protein tyrosine kinase activity of the EGF receptor in intact cells, suggesting substrate specificity in vivo.

Antigens, CD↗
PubMed1990

[Medial coxa saltans--a difficult diagnosis].

A case of medial coxa saltans is presented here. This is a very rare condition and very difficult to diagnose, which is illustrated by the case history. Bursography as a diagnostic aid is described and a possible form of operative treatment of the condition is reviewed.

Adolescent↗
PubMed1988

Cloned cytotoxic T lymphocyte target cells fail to induce early activation events in effector cytotoxic T lymphocytes.

We have explored further the basis for resistance of cloned cytotoxic T lymphocytes (CTLs) to cell-mediated cytotoxicity. We find that most cloned CTLs recognized as specific target cells by other cloned CTLs used as effector cells fail to activate three early events that may be critical in triggering lysis in the effector CTLs: Ca2+ influx, microtubule organizing center (MTOC) reorientation, and serine esterase release. To the extent that any or all of these events are involved in activation or expression of the lytic pathway in effector CTLs, our results suggest that in addition to being inherently resistant to cytotoxic granule extracts, many CTLs are also unable to induce lytic function in other (effector) CTLs. We have found one CTL clone that can respond to recognizable cloned CTL target cells with at least MTOC reorientation and serine esterase release, although the target CTLs are still not lysed. In this case, the resistance of the target CTL to lysis may be due solely to its resistance to cytoplasmic granule contents.

Animals↗
PubMed1987

The role of Ca2+ in activation of mature cytotoxic T lymphocytes for lysis.

We carried out a detailed analysis of the requirement for Ca2+ in the lysis of target cells by cloned cytotoxic T lymphocytes (CTL). In direct, antigen-specific lysis we always observed an influx of Ca2+ into the CTL concomitant with target cell binding. However, we never observed an increase in CTL Ca2+ content during lectin-mediated lysis, or nonspecific lysis by phorbol myristate acetate-induced CTL. We found that in all three types of lysis (direct, lectin-mediated lysis, C or phorbol myristate acetate-induced) the requirement for Ca2+ in lysis was dictated by the target cell used; the same CTL can kill one target cell in the absence of detectable Ca2+, and absolutely require Ca2+ for the lysis of another target cell. Target cell killing, when it occurred in the absence of Ca2+, was accompanied by microtubule organizing center reorientation in the CTL, showing that this function is not uniformly Ca2+ dependent. These results provide further evidence that Ca2+ is not always required for activation of the lytic pathway in CTL, although Ca2+ may be absolutely required for other CTL functions such as interleukin production or expression of the interleukin 2 receptor.

Animals↗
PubMed1987

Resistance of cloned cytotoxic T lymphocytes to cell-mediated cytotoxicity.

Cloned CTLs show an unusually high resistance to lysis by effector CTLs. Several cloned CTL lines in our laboratories are absolutely refractory to lysis by other cloned CTLs, either (a) directly, (b) in the presence of lectin, or (c) by PMA-induced CTLs. They can be lysed to some extent by primary CTL, although they are less than 5% as sensitive as target cells normally used to assay primary CTL lytic activity. Lysis of cloned CTLs by primary CTL effector cells is not enhanced by the presence of lectin, and cloned T cells are also highly resistant to lysis by primary lymphokine-activated killer cells. Cloned CTLs are highly resistant to lysis by isolated CTL granules that contain the membranolytic pore-forming protein (PFP or perforin), while non-CTL targets are highly susceptible to granule-mediated killing, indicating that cloned CTLs resist lysis not only at the intact effector cell level but also when soluble effector proteins are used. This resistance mechanism may explain how CTLs kill but spare themselves from being killed during the cytolytic event.

Animals↗