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W H Fischer

Publications and source records attributed to W H Fischer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Activin and inhibin binding to the soluble extracellular domain of activin receptor II.

Activins and inhibins belong to the transforming growth factor-beta-like superfamily of growth and differentiation factors that exert pleiotropic effects in many target tissues. Heteromeric association of activin with two structurally related receptor serine/threonine kinases, activin receptor types I and II, initiates downstream signaling events. The extracellular domain of type II mouse activin receptor (ActRII ECD) was expressed in the baculovirus system, purified in three steps by lectin affinity, anion exchange, and reverse phase chromatography, and further characterized by mass spectrometry. The reduction in the apparent size of the purified ActRII ECD on SDS-PAGE after treatment with glycosidases provided evidence for N- and O-linked oligosaccharides. Specific receptor/ligand complexes of [125I] activin A to ActRII ECD or [125I]ActRII ECD to activin A were analyzed by cross-linking and immunoprecipitation. Two major radiolabeled bands were observed on SDS-PAGE with mobilities consistent with the expected size of ActRII ECD/betaA or ActRII ECD/betaAbetaA. When inhibin A was cross-linked to [125I]ActRII ECD, a slower migrating complex corresponding to ActRII ECD/betaAalpha was also observed. The apparent dissociation constant (Kd) for activin A binding to ActRII ECD was 2-7 nM. This Kd value is approximately an order of magnitude greater than that of the full-length membrane-associated type II receptor. Treatment of cultured rat anterior pituitary cells with ActRII ECD attenuated FSH secretion in response to exogenous activin A or endogenous activin B. These data indicate that the soluble ActRII ECD has structural determinants that are sufficient for high affinity ligand binding.

Activin Receptors↗
PubMed1998

Influence of diet restriction and tumor promoter dose on cell proliferation, oxidative DNA damage and rate of papilloma appearance in the mouse skin after initiation with DMBA and promotion with TPA.

The mouse skin tumor initiation-promotion model was used to investigate the protective effect of diet restriction in mechanistic and quantitative terms. A total of five groups of 14 male NMRI mice were initiated with 100 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly with 2.5, 1.25, or 0.625 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). Food intake was ad libitum (all 3 TPA dose levels) or restricted to 70% (high and intermediate TPA dose levels). Time of appearance of the first papilloma was recorded for each mouse. Two weeks later, an osmotic minipump delivering 5-bromo-2'-deoxyuridine (BrdU) was implanted and the mouse was killed after 24 h. Cell proliferation in the epidermis was assessed by immunohistochemistry for BrdU incorporated into DNA. 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in epidermal DNA was determined by HPLC/electrochemical detection. The median latency time (t50) for the appearance of skin papilloma in the high-, intermediate-, and low-dose TPA groups fed ad libitum was 9, 15.5, and 23.5 weeks, respectively. The diet-restricted groups (high and intermediate TPA dose) showed t50 values of 16 and 26 weeks. Therefore, diet restriction to 70% had approximately the same protective effect as reducing the dose of TPA by a factor of two. Both the rate of cell proliferation and the level of 8-OH-dG in the epidermis increased with the dose of TPA. Median values were increased 3- to 4-fold at the highest dose. In controls, but not in TPA-treated animals, diet restriction resulted in a decrease for both markers, by 25 and 40% for the labeling index for cell division and the level of 8-OH-dG, respectively. Both markers showed an inverse relationship with the median papilloma latency time. On an individual basis, the correlation was significant in some groups, but only for the labeling index. The data indicate that protection from the skin tumor-promoting effect of TPA by diet restriction could be based more on a reduction of the rate of cell division than on a reduction of oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.

The HIV-1 Tat protein regulates transcription elongation through binding to the viral TAR RNA stem-loop structure. We have isolated a novel 87 kDa cyclin C-related protein (cyclin T) that interacts specifically with the transactivation domain of Tat. Cyclin T is a partner for CDK9, an RNAPII transcription elongation factor. Remarkably, the interaction of Tat with cyclin T strongly enhances the affinity and specificity of the Tat:TAR RNA interaction, and confers a requirement for sequences in the loop of TAR that are not recognized by Tat alone. Moreover, overexpression of human cyclin T rescues Tat activity in nonpermissive rodent cells. We propose that Tat directs cyclin T-CDK9 to RNAPII through cooperative binding to TAR RNA.

3T3 Cells↗
PubMed1998

Assembly of proteins to postsynaptic densities after transient cerebral ischemia.

Transient ischemia leads to changes in synaptic efficacy and results in selective neuronal damage during the postischemic phase, although the mechanisms are not fully understood. The protein composition and ultrastructure of postsynaptic densities (PSDs) were studied by using a rat transient ischemic model. We found that a brief ischemic episode induced a marked accumulation in PSDs of the protein assembly ATPases, N-ethylmaleimide-sensitive fusion protein, and heat-shock cognate protein-70 as well as the BDNF receptor (trkB) and protein kinases, as determined by protein microsequencing. The changes in PSD composition were accompanied by a 2.5-fold increase in the yield of PSD protein relative to controls. Biochemical modification of PSDs correlated well with an increase in PSD thickness observed in vivo by electron microscopy. We conclude that a brief ischemic episode modifies the molecular composition and ultrastructure of synapses by assembly of proteins to the postsynaptic density, which may underlie observed changes in synaptic function and selective neuronal damage.

Adenosine Triphosphatases↗
PubMed1998

Microsequencing of bovine cerebrospinal fluid apolipoproteins: identification of bovine apolipoprotein E.

In studies of bovine plasma lipoproteins, apolipoprotein E (apoE) was not found associated with alpha-lipoproteins isolated over a broad range of densities. However, studies of cerebrospinal fluid (CSF) lipoproteins from other mammals have shown that apoE is a major apolipoprotein associated with high density lipoprotein, a fact that prompted us to determine if this were also the case in bovine CSF. CSF samples were obtained from animals with a surgically implanted catheter. Most analyzed samples were obtained from cows at various stages of the postpartum period; however, a few samples also were obtained at term or during pregnancy. Analyses of isolated ultracentrifugal fractions by polyacrylamide gel electrophoresis revealed the presence of two apo, with the expected molecular weights for apoE and apoA-I. By using both matrix-assisted laser desorption mass spectrometry and microsequencing techniques, we demonstrated that these apo are indeed apoE and apoA-I.

Amino Acid Sequence↗
PubMed1997

Regulation of B cell functions by C3a and C3a(desArg): suppression of TNF-alpha, IL-6, and the polyclonal immune response.

Regulation of the humoral immune response by fragments of complement component C3 has been shown to play an important role in host defense. In this study, we investigated expression of the C3a receptor (C3aR) by human B cells and the effects of C3a and C3a(desArg) on IgG, TNF-alpha, and IL-6 production in Staphylococcus aureus Cowan strain I (SAC)/IL-2-activated B cells. Here we report that tonsil-derived human B cells express a C3aR that is indistinguishable at both the protein (by flow cytometry) and mRNA (by reverse transcription-PCR) levels from that on human neutrophils or on C3aR-transfected mouse L cells. Incubation of SAC/IL-2-activated B cells with C3a or C3a(desArg) resulted in a dose-dependent suppression of the polyclonal immune response. Under these same experimental conditions, IL-6 and TNF-alpha release was suppressed in a dose-dependent manner. Kinetic studies with SAC/IL-2-activated B cells revealed that C3a must be present at initiation of the culture to exert its suppressive effect on IgG and IL-6 production. These results demonstrate that tonsil-derived B cells express the C3aR and that C3a as well as C3a(desArg) have a direct immunomodulatory effect on these cells.

Adolescent↗
PubMed1997

Sequence of two gonadotropin releasing hormones from tunicate suggest an important role of conformation in receptor activation.

The primary structure of two forms of gonadotropin releasing hormone (GnRH) from tunicate (Chelyosoma productum) have been determined based on mass spectrometric and chemical sequence analyses. The peptides, tunicate GnRH-I and -II, contain features unprecedented in vertebrate GnRH. Tunicate GnRH-I contains a putative salt bridge between Asp5 and Lys8. A GnRH analog containing a lactam bridge between Asp5 and Lys8 was found to increase release of estradiol compared with that of the native tunicate GnRH-I and -II. Tunicate GnRH-II contains a cysteine residue and was isolated as a dimeric peptide. These motifs suggest that the conformation plays an important role in receptor activation.

Amino Acid Sequence↗
PubMed1997

Regulation of the ERBB-2 promoter by RBPJkappa and NOTCH.

Within the human ERBB-2 gene promoter, a 100-base pair region 5' to the TATA box enhances basal transcription 200-fold. Two palindromes present within this 100-base pair region are important for transcription. The palindrome binding protein was purified to homogeneity and found to be identical to RBPJkappa, the mammalian homolog of Drosophila Suppressor of Hairless (Su(H)). Recombinant RBPJkappa bound the ERBB-2 promoter with affinity comparable with that seen with well characterized RBPJkappa binding sites. RBPJkappa activated an ERBB-2 palindrome-containing promoter in 293 cells. Because in Drosophila Su(H) acts downstream of NOTCH and because NOTCH.Su(H)/RBPJkappa stimulates transcription from target promoters, NOTCH-IC, a constitutively active form of NOTCH, was tested for effects on the ERBB-2 palindrome. NOTCH-IC further increased RBPJkappa-mediated transcription on wild type but not mutant ERBB-2 palindrome. Thus, RBPJkappa can activate ERBB-2 transcription and serve as an anchor to mediate NOTCH function on the ERBB-2 gene.

Animals↗
PubMed1997

A cellular cofactor for the constitutive transport element of type D retrovirus.

A human nuclear protein that specifically interacts with the constitutive transport element (CTE) of simian retrovirus was identified as adenosine 5'-triphosphate-dependent RNA helicase A. This protein could bind to functional CTE but not to inactive CTE mutants. The interaction of helicase A with CTE was distinct from previously described helicase activity of this protein. Helicase A shuttled from the nucleus to the cytoplasm in the presence of a transcription inhibitor or in cells transiently overexpressing CTE-containing RNA. In vivo colocalization of helicase A and CTE was observed in experiments that combined in situ hybridization and immunostaining. These results suggest that helicase A plays a role in the nuclear export of CTE-containing RNA.

Adenosine Triphosphate↗
PubMed1997

Primary structure of three forms of gonadotropin-releasing hormone (GnRH) from the pacu brain.

Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu-His-Trip-Ser-Tyr-Gly-Leu-Ser -Pro-Gly-NH2, 1113.4 Da); chicken GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2, 1236.6 Da); and salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2, 1212.3 Da). In addition the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.

Amino Acid Sequence↗
PubMed1997

Determination of phosphorylation sites in peptides and proteins employing a volatile Edman reagent.

A manual Edman degradation protocol has been developed that allows the identification of phosphorylation sites in 32P-labeled peptides at the subpicomole level. By using both a volatile reagent, trifluoroethyl isothiocyanate, and volatile buffers, extraction steps are rendered unnecessary and cycle times can be reduced to 45 min. The protocol was employed to identify the site of phosphorylation in phosphoserine- and phosphotyrosine-containing peptides.

Binding Sites↗
PubMed1997

Evidence that gonadotropin-releasing hormone (GnRH) functions as a prolactin-releasing factor in a teleost fish (Oreochromis mossambicus) and primary structures for three native GnRH molecules.

Three forms of gonadotropin-releasing hormone (GnRH) are isolated and identified here by chemical sequence analysis for one species of tilapia, Oreochromis niloticus, and by HPLC elution position for a second species of tilapia, O. mossambicus. Of the three GnRH forms in O. mossambicus, chicken GnRH-II (cGnRH-II) and sea bream GnRH (sbGnRH) are present in greater abundance in the brain and pituitary than salmon GnRH (sGnRH). These three native forms of GnRH are shown to stimulate the release of prolactin (PRL) from the rostral pars distalis (RPD) of the pituitary of O. mossambicus in vitro with the following order of potency: cGnRH-II > sGnRH > sbGnRH. In addition, a mammalian GnRH analog stimulated the release of PRL from the pituitary RPD incubated in either iso-osmotic (320 mosmol/l) or hyperosmotic (355 mosmol/l) medium, the latter normally inhibiting PRL release. The response of the pituitary RPD to GnRH was augmented by co-incubation with testosterone or 17 beta-estradiol. The effects of GnRH on PRL release appear to be direct effects on PRL cells because the RPD of tilapia contains a nearly homogeneous mass of PRL cells without intermixing of gonadotrophs. Our data suggest that GnRH plays a broad role in fish, depending on the species, by affecting not only gonadotropins and growth hormone, but also PRL.

Amino Acid Sequence↗
PubMed1996

Two new forms of gonadotropin-releasing hormone in a protochordate and the evolutionary implications.

The neuropeptide gonadotropin-releasing hormone (GnRH) is the major regulator of reproduction in vertebrates. Our goal was to determine whether GnRH could be isolated and identified by primary structure in a protochordate and to examine its location by immunocytochemistry. The primary structure of two novel decapeptides from the tunicate Chelyosoma productum (class Ascidiacea) was determined. Both show significant identity with vertebrate GnRH. Tunicate GnRH-I (pGlu-His-Trp-Ser-Asp-Tyr-Phe-Lys-Pro-Gly-NH2) has 60% of its residues conserved, compared with mammalian GnRH, whereas tunicate GnRH-II (pGlu-His-Trp-Ser-Leu-Cys-His-Ala-Pro-Gly-NH2) is unusual in that it was isolated as a disulfide-linked dimer. Numerous immunoreactive GnRH neurons lie within blood sinuses close to the gonoducts and gonads in both juveniles and adults, implying that the neuropeptide is released into the bloodstream. It is suggested that in ancestral chordates, before the evolution of the pituitary, the hormone was released into the bloodstream and acted directly on the gonads.

Amino Acid Sequence↗
PubMed1996

Characterization of a sheep brain corticotropin releasing factor binding protein.

We report here the identification, purification and cDNA cloning of a corticotropin releasing factor (CRF) binding protein(s) (CRF-BP) from sheep brain. Native sheep and rat brain CRF-BP and recombinant rat CRF-BP were shown to be N-glycosylated. Two membrane associated forms of brain CRF-BPs of 33 and 35 kDa were purified from sheep brain homogenates after solubilization in the presence of detergent. N-Terminal sequence analysis revealed that the 35 kDa protein is proteolytically cleaved near the N-terminus giving rise to an 18 amino acid peptide and a 33 kDa CRF-BP. Both the purified 33 and 35 kDa ovine CRF-BPs could be specifically cross linked to ovine [125I]CRF and human [125I]CRF. In contrast, recombinant rat CRF-BP can only be cross-linked to human [125I]CRF. A 1.7 kb cDNA clone (Basil 7) encoding an open reading frame for a 324 amino acid CRF-BP precursor was cloned from a sheep brain lambda gtlO cDNA library and was shown to have 85% and 87% amino acid homology to the rat and human proteins, respectively. Competitive binding analysis of the recombinant sheep CRF-BP (Basil 7) expressed in CHO cells revealed that it binds human and ovine CRF with high affinity. However, the recombinant sheep CRF-BP (Basil 7) had approximately 50-fold higher affinity for human CRF than for the ovine peptide. These data present the first biochemical proof that CRF-BP is in the brain and provides evidence for the existence of different forms of CRF-BP which have evolved across species to regulate CRF.

Amino Acid Sequence↗
PubMed1996

A role for CD81 in early T cell development.

Early stages of T cell development are thought to include a series of coordinated interactions between thymocytes and other cells of the thymus. A monoclonal antibody specific for mouse CD81 was identified that blocked the appearance of alpha beta but not gamma delta T cells in fetal organ cultures initiated with day 14.5 thymus lobes. In reaggregation cultures with CD81-transfected fibroblasts, CD4-CD8- thymocytes differentiated into CD4+CD8+ T cells. Thus, interactions between immature thymocytes and stromal cells expressing CD81 are required and may be sufficient to induce early events associated with T cell development.

Amino Acid Sequence↗
PubMed1996

Dose-time response in mouse skin tumor induction by 7, 12-dimethylbenz[a]anthracene and 12-O-tetradecanoyl-phorbol-13-acetate.

The question was addressed whether the dose-response relationship derived from a carcinogenicity study can be used for mechanistic interpretation and to what extent the shape of the curve is dependent on the duration of the bioassay and the time of analysis. The mouse skin tumor model was used. It allows recording of the time of tumor appearance without interim sacrifice. Groups of 16 female NMRI mice were treated twice weekly by dermal administration with combinations of (a) 2.5 nmol 12-O-tetradecanoyl-phorbol-13-acetate (TPA) plus 0, 0.3, 1, 3, or 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) or with (b) 2.5 nmol DMBA plus 0, 0.1, 0.3, 1, 3, or 10 nmol TPA. The appearance of the first papilloma was recorded for each animal and the cumulative incidence data were analyzed in two ways: (i) With the usual dose-prevalence representation at a fixed time point, the dose response for TPA was sigmoidal, while it was linear-superlinear for DMBA. This was observed at all time points, indicating that the dose-response information may be used for a distinction between DNA-reactive and tumor-promoting mechanisms of action. (ii) When time-to-tumor and loss of tumor-free lifetime was analyzed as a function of dose, there was again a marked difference between DMBA and TPA. The tumor-free lifetime increased with each step of dose reduction but the slope was about four times larger for TPA compared with that for DMBA. Further reduction of the TPA dose could result in a situation in which the natural life span sets a limit to an observable effect. Under the conditions of this bioassay for mouse skin papilloma induction by a combination treatment with DMBA plus TPA, the findings support the idea of a no-effect low-dose threshold for the tumor-promoting agent.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Response properties of relay cells in the A-laminae of the cat's dorsal lateral geniculate nucleus after saccades.

Responses of relay cells in the A-laminae of the dorsal lateral geniculate nucleus (LGNd) during spontaneous saccades and saccade-like visual stimulation were extracellularly recorded in awake cats. Ninety-six out of 137 cells recorded (42 X and 54 Y cells) were responsive during spontaneous saccadic eye movements. All Y cells and 67% of the X cells responded with burst activity, i.e. with either one or two activity peaks during and after saccades. Thirty-three percent of the X cells were inhibited during saccades. Excitatory peaks occurred at mean latencies of 33 ms and 31 ms for X and Y cells, respectively. Comparable burst responses were obtained when retinal image shifts similar to those during saccades were induced by external saccade-like stimulus movements. However, the latencies of excitatory peak activity were significantly longer to external stimuli than to the onsets of saccades. This indicates the existence of an eye movement-related input which activates LGNd relay cells in addition to the visual input. We propose that the pretectogeniculate projection may contribute to the responses of LGNd relay cells following saccadic eye movements via a disinhibitory input and that this input could be involved in intra- and postsaccadic modulations of the transfer of visual signals to visual cortex.

Animals↗