A chimeric tyrosine/tryptophan hydroxylase

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t_1/2 of 84 min at 37°C, TPH was much less stable (t _1/2=28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by at _1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.     

Fear Imagery and Text Processing

This study examined the effect of three variables held to influence heart rate response during imagery-related text processing: mode of processing, content of text, and inclusion of response information in the text. Sixty-four undergraduates imagined and silently repeated fearful and neutral sentences in a paradigm designed to allow for self-initiation of sentence processing. Fear sentences either included or did not include information about bodily responses in the image. Heart rate accelerated more during fear imagery than during neutral imagery or silent repetition of either type of sentence. Inclusion of response information in fear material did not increase heart rate response to imagery, but did affect self-report in the predicted direction. Heart rate waveform prior to the sentence tasks indicated pre-processing of fearful material. The results were discussed in relation to a bio-informational theory of imagery, which asserts that emotional imagery accesses the same centrally-mediated response program as is evoked in the target reality context, and thus occasions measurable activity in the appropriate effectors.     

Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21–41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (NΔ15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion NΔ15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (NΔ30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (NΔ41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.     

Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques [see comments]

Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.     

The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils

The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface- bound plasminogen to plasmin, a process of presumed importance for a number of biologic processes including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor- alpha results in a rapid increase in the expression of uPAR. This process is accompanied by an increased cell-associated plasminogen activation after preincubation of neutrophils with pro-uPA in vitro. By subcellular fractionation of unstimulated neutrophils, 50% of uPAR is recovered in fractions containing latent alkaline phosphatase, corresponding to an intracellular compartment of easily mobilizable secretory vesicles distinct from both primary and specific granules, whereas the remaining 50% of uPAR is associated with a compartment eluting close to the specific granules. In contrast, the ligand pro-uPA is primarily (approximately 80%) found in the specific granules, but small amounts of pro-uPA/uPA (approximately 20%) coelute with latent alkaline phosphatase. Stimulation of neutrophils with FMLP results in translocation of uPAR as well as of pro-uPA from the secretory vesicles, whereas stimulation with PMA is required to translocate material from specific granules. Flow cytometry of neutrophils saturated with exogenous diisopropyl fluorophosphate-uPA shows a large excess (approximately 90%) of unoccupied uPAR on resting as well as FMLP- and PMA-stimulated neutrophils, suggesting a possible role for exogenous pro-uPA in providing neutrophils with a potential for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi.     

A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation

P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG₁) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M_r) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIbα). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of ¹²⁵I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825±2886, mean ±SD) (K_d=1.5±0.5 nm ) compared to resting platelets (2801±1278, mean ±SD) (K_d=1.5±0.6 nm ), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG₁, F(ab′)₂ or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf ) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell–cell contact, may play an active role in platelet–platelet interactions.     

The future of proteomics in the study of alcoholism

This article represents the proceedings of a workshop at the 2003 annual meeting of the Research Society on Alcoholism in Fort Lauderdale, FL. The workshop organizers/chairpersons were Chinnaswamy Kasinathan and Paul Manowitz. The presentations were (1) Introduction to the field of proteomics, by Kent Vrana; (2) Use of proteomics in the identification of urinary biomarkers for alcohol intake, by Chinnaswamy Kasinathan, Paul Thomas, and Paul Manowitz; (3) Proteomics screening illuminates ethanol-mediated induction of HDL proteins in macaques, by Kent Vrana, Randy Gooch, Travis Worst, Stephen Walker, Aaron Xu, Peter Pierre, Heather Green, and Kathleen Grant; and (4) Proteomics applied to the study of the liver, by Laura Beretta.     

Evidence that large granular lymphocytes from B-CLL patients with hypogammaglobulinemia down-regulate B-cell immunoglobulin synthesis [published erratum appears in Blood 1989 Jun;73(8):2232]

B-chronic lymphocytic leukemia (CLL) patients frequently suffer from moderate to severe hypogammaglobulinemia. This complication is a serious cause of morbidity and mortality in this disorder. There is recent evidence that natural killer (NK) cells modulate B-cell immunoglobin (Ig) synthesis/secretion. The authors therefore evaluated the circulating NK cells from B-CLL patients on their ability to regulate mitogen-induced B-cell Ig synthesis. Blood, NK cells (CD16+, CD3-) from three B-CLL patients with hypogammaglobulinemia were able to clearly down-regulate the pokeweed mitogen (PWM)-induced-B-cell Ig secretion. In contrast, CD16+, CD3- cells from age-sex-matched controls or B-CLL patients with normal Ig were either nonregulatory or enhanced mitogen-induced B-cell Ig secretion. An alternative explanation for hypogammaglobulinemia in B-CLL patients is the immunomodulation of B- cell Ig production/secretion by CD16+, CD3- blood cells.     

The effect of healing phenotype-inducing cytokine formulations within soft hydrogels on encapsulated monocytes and incoming immune cells

The adverse immune responses to implantable biomedical devices is a general problem with important consequences for the functionality of implants. Immunomodulatory soft hydrogel-based interfaces between the implant and the host can attenuate these reactions. Moreover, encapsulation of the patient''s own immune cells into these interfaces can lead to the personalisation of implants from the immune reaction point of view. Herein, we described a co-crosslinkable composite hydrogel (composed of gelatin and hyaluronic acid), which could be used for the encapsulation of macrophages in the presence of an anti-inflammatory phenotype-fixing cytokine cocktail. To mimick the incoming immune cells on the coating surface in vivo, peripheral blood mononuclear cells were seeded on the hydrogels. The encapsulation of monocytic cells into the composite hydrogels in the presence of cytokine cocktails at 5× or 10× concentrations led to the spreading of the encapsulated cells instead of the formation of clusters. Moreover, the secretion of the anti-inflammatory cytokines IL-1RA and CCL-18 was significantly increased. The attachment of PBMC to the surface of the hydrogel is dependent on the hydrogel composition and also significantly increased in the presence of the cytokine cocktail together with the number of CD68+ cells on the hydrogel surface. Our study demonstrates that the delivery of a polarisation cocktail with biocompatible hydrogels can control the initial response by the incoming immune cells. This effect can be improved by the encapsulation of autologous monocytes that are also polarised by the cytokine cocktail and secrete additional anti-inflammatory cytokines. This interface can fine tune the initial immune response to an implanted biomaterial in a personalised manner.

Hydrogels made from the derivatives of gelatin and hyaluronic acid were used as coatings to control the immune responses.