Functional association of TGF-beta receptor II with cyclin B

Utilizing the cytoplasmic tail of Transforming Growth Factor Receptor Type II (TGFbeta RII) as bait in a yeast two hybrid system, we have identified human cyclin B2 as a direct physical partner of TGFbeta RII. Analysis of deletion mutants of glutathione-S-transferase (GST)-cyclin B2 mapped its binding domain for TGFbeta RII to the C-terminal and revealed a negative regulatory region immediately upstream of the cyclin box. Using recombinant proteins, Cdc2 was demonstrated to indirectly interact with TGFbeta RII via cyclin B2. This interaction was reproduced in THP-1 monocytic cells, where TGFbeta treatment markedly enhanced the ability of cyclin B2 and, correspondingly, Cdc2 from TGFbeta-treated THP-1 cells, to bind the GST-TGFbeta RII fusion protein. More importantly, TGFbeta RII co-precipitated with cyclin B2 in TGFbeta-treated THP-1 cells. TGFbeta treatment also caused threonine phosphorylation of Cdc2 in the TGFbeta RII-cyclin B2-Cdc2 complex in THP1 cells, in parallel with down regulation of Cdc2 function as measured by histone H1 kinase activity. Cyclin B1 had the same capacity to bind TGFbeta RII and mediate indirect Cdc2 binding. These results suggest an alternative mechanism that cell cycle arrest in the G1/S phase caused by TGFbeta may, in part, be due to inactivation of cyclin B/Cdc2 kinase, which is needed for entry into the G2/M phase.     

Isolation of MHC class I-restricted tumor antigen peptide and its precursors associated with heat shock proteins hsp70, hsp90, and gp96

We have previously demonstrated that vaccination with heat shock proteins hsp70, hsp90, and gp96 elicits specific immunity against the tumor from which the hsps were purified. Although the association of tumor Ag peptides with these hsps have been suggested, the identification of the peptides or their precursors stripped from the hsps remained to be resolved. We show in this report that an Ld-restricted cytotoxic T lymphocyte epitope of a mouse leukemia RLmale symbol1 and its precursors are associated with the chaperones hsp90 and hsp70 in the cytosol and gp96 in the lumen of the endoplasmic reticulum. Hsp70 was associated with only final sized octamer, while hsp90 was found to associate with the octamer and two distinct precursor peptides. The gp96 was associated with the octamer and one of the two precursors. Thus, each of the hsps bound a distinct set of peptides. Our results have demonstrated for the first time that the hsps associate not only with final sized tumor Ag peptide but also with its precursors. The implication of this evidence is also discussed in terms of the roles of hsps in MHC class I Ag processing/presentation.     

An Investigation of the Dynamic Response of Thermocouples in Inert and Reacting Condensed Phase Energetic Materials

Thermocouples are widely used to measure both transient and steady state temperatures in many different materials. Bead diameters range from a few micrometers to several millimeters. During steady state operations, dynamic response is not an issue. However, during fluctuating conditions, the temporal response of the thermocouple must be taken into account. Several different techniques have been developed to measure the response characteristics of measurements in inert gases and liquids, but to the authors' knowledge, relatively little work has been conducted in condensed phase reacting materials. This report presents an initial survey of the topic.     

Modification of ventricular tachycardia by carotid sinus massage

Imiquimod and its analogs belonging to a class of imidazoquinolinamines, activate immune system via cytokine induction, and have antitumor and antiviral effects in mammals. In this study, we showed that a related analog, designated S-28828, induced interferon (IFN) and macrophage activating cytokine(s) (macrophage activating factor, MAF) in chickens in vivo, ex vivo, and in vitro. IFN and MAF were detectable in the serum of chickens following oral administration. Serum IFN levels were the highest at 2 h after treatment. Although there was no detectable IFN in sera of chickens at 8, 24, and 48 h after treatment, high levels of interferon inducible enzyme, 2'-5' oligoadenylate synthase (2'5'OAS) were present at these time points. In vitro and ex vivo studies showed that spleen cells, bone marrow (BM) cells, and peripheral blood leukocytes (PBL) were capable of producing IFN and MAF, although spleen cells produced the highest levels. Our results suggest that S-28828 administered orally may be a useful immunoenhancing and antiviral agent for chickens.     

Maxillary metastasis of osteosarcoma

A retrospective study of carcinomas of the hypopharynx was conducted to examine the epidemiological and clinical features of this cancer in Senegal. An analysis of 66 cases showed a very particular distribution in Senegal with more female (57.6%) than male cases. Average age at diagnosis was 33 years. Advanced cancer (T3-T4) was noted in 65% of the cases, but the first physical examination noted the absence of cervical nodes in 53.6% of the cases. Location at the piriform sinus was observed in 41% of the cases, followed by the posterior wall of the hypopharynx (25.7%). Chronic anemia with iron deficiency was seen in 30% of the cases, probably an important etiological factor of the cancer in Senegal. This point would be the goal of a future prospective study.     

Effects of endurance training in the leopard shark, Triakis semifasciata

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an alpha-helix content of approximately 42%. The alpha-helix comprises an uninterrupted hydrophobic stretch of < or = 12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15-75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10(-4) seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed.     

N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.