Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.     

A new in vitro system for studying cell response to mechanical stimulation : Different effects of cyclic stretching and agitation on smooth muscle cell biosynthesis

An experimental model has been devised to permit morphologic and metabolic characterization of cells subjected to a range of cyclic mechanical stimuli similar to those which may prevail in blood vessel walls. A unique feature is the use of purified elastin membranes prepared from bovine aortas as extensible substrates for cell growth. Cells attached firmly to such membranes which could then be subjected to continuous stretching and relaxation or displacement without stretching by a motor coupled to a movable supporting frame. Various combinations of frequencies, amplitudes and rates of deformation have been used over extended periods with minimal fatigue or disruption of the elastin substrate. The effects of cyclic stretching on [¹⁴C]proline incorporation into protein and collagen and [³H]thymidine incorporation into DNA by rabbit aortic smooth muscle cells were distinct from those attributable to agitation without stretching, indicating that cells responded differently to these modes of stimulation. Increases in rate of protein or DNA synthesis induced by stretching were just as marked after 48 h of stimulation as they were at the outset of an experimental period. Since the system permits observations of cell response to independently variable components of pulsatile stress over extended periods and under a variety of culture conditions, it may be expected to provide new data concerning the interaction of mechanical with hormonal and genetic factors in the elaboration of connective tissue components.     

Tumor-associated antigens of rat moloney sarcoma cells. II. Cytosol antigens.

Rat Moloney sarcoma cells (MST) were pulsed with 35S-L-methionine for 10 and 60 min and lysed by vortexing in 0.5% deoxycholate, 0.5% NP40, 0.02 M Tris, 0.05 M NaCl, pH 7.5, for 30 sec. The lysate was centrifuged at 16,300 X G for 10 min and the supernatant was co-precipitated with Ig fractions of normal BN serum, normal Lewis serum, BN antiserum to Moloney sarcoma cells (BNaMST), BN antiserum to tumor-associated antigens (BNaTAA), BN antiserum to murine leukemia virus (BNaMuLV), BN antiserum to p30 (BNap30), BN antiserum to gp70 (BNagp70), Lewis antiserum to BN (LeaBN), and BN antiserum to BC5 tumor (BNaBC5). With BNaTAA and BNaMST, a cytoplasmic TAA with m.w. 85,000 was detected. In addition, BNaTAA detected three other species of cytoplasmic TAA with m.w. 220,000, 170,000 and 39,000.     

Transformation and allelic replacement in Francisella spp.

We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis

Coriolus versicolor (CV), also known as Yunzhi, is one of the commonly used Chinese medicinal herbs. Although recent studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized aqueous ethanol extract prepared from Coriolus versicolor on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. Cell death ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis. The present results demonstrated that CV extract at 50 to 800 microg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90% (p < 0.01), with ascending order of IC50 values: HL-60 (147.3 +/- 15.2 microg/ml), Raji (253.8 +/- 60.7 microg/ml) and NB-4 (269.3 +/- 12.4 microg/ml). The extract however did not exert any significant cytotoxic effect on normal liver cell line WRL (IC50 > 800 microg/ml) when compared with a chemotherapeutic anticancer drug, mitomycin C (MMC), confirming the tumour-selective cytotoxicity. Nucleosome productions in HL-60, NB-4 and Raji cells were significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.     

Pattern recognition analysis to the variation of nasal-pharynx cancer patients' trace element levels in samples of hair,whole blood,and tissue

Pattern recognition has been used in this paper to analyze trace element levels in patients diagnosed with nasal-pharynx cancer (NPC) and in healthy control subjects. Trace elements such as Zn, Cu, Fe, Mn, and Mg have been tested in samples of hair and whole blood. Through Mahalanobis Distance Decision analysis, we have achieved good classification effects in whole blood samples: Efficiency for distinguishing patients is 96% and that of healthy controls is 90%. Classification hair samples is inferior to whole blood: Decision accuracy for patients is 58% and healthy controls is 90%. These results are also shown in a nonlinear mapping figure. At the same time, we have also determined 5 trace element levels in 16 other cancer patients' nonneoplastic and cancerous tissue, with no significant difference between them: Decision accuracy of cancerous tissue is 63%, and in nonneoplastic tissue is 50%, hence, we cannot identify them. It can be inferred that there is no idiosyncratic change of trace elements in cancer patients' neoplastic tissue, the change of a cancerous person may occur in the whole body.     

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.