Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

Bullous pemphigoid antigen. II. Isolation from the urine of a patient.

This report concerns the isolation of bullous pemphigoid antigen from the nondialyzable urinary components of a patient with the disease. The isolation was accomplished by ion exchange chromatography and gel filtration. Pemphigoid antigen was found to be a basic glycoprotein that on SDS gel electrophoresis showed two major bands, one in the 18,000 m. w. region and the second with a m. w. of 74,000. Between these two bands, two additional bands appeared; one of 35,000 daltons and the other of 68,000 daltons. The 18,000 m. w. band was eluted from the gel and rerun on SDS gels. These gels showed the 18,000 m.w. band and also the appearance of the 35,000 and 74,000 m.w. bands. This finding indicates that urinary pemphigoid antigen may exist both as a single monomeric form and in polymeric aggregates.     

(14C)acetate assimilation by a type I obligate methylotroph, Methylococcus capsulatus.

Methanol and formate oxidation supported the assimilation of [14C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-14C]acetate assimilation. The amount of [1-14C]acetate assimilation supported by methanol oxidation was significantly inhibited by cyanide.     

Lactose-proton symport by purified lac carrier protein

The lac carrier protein of Escherichia coli was purified by an improved procedure and its activity assayed by a rapid filter method. Following reconstitution of the carrier by octyl glucoside dilution, proteoliposomes were concentrated by filtration on a microporous filter. Lactose accumulation by adsorbed or entrapped proteoliposomes is driven by an artificially imposed pH gradient (interior alkaline), by a membrane potential (interior negative), or by a combination of both forces. Activity is almost completely abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone or by the competitive inhibitor thiodigalactoside. Addition of lactose to proteoliposomes under appropriate conditions results in alkalinization of the external medium. This effect is not observed with liposomes devoid of lac carrier or in the presence of proton conducting agents. The results provide a strong indication that the lac gamma gene product is the only protein in the cytoplasmic membrane of Escherichia coli required for lactose-proton symport.     

A complex pattern of H2A phosphorylation in the mouse testis

Phosphorylation of H2A histones in mouse testis was examined using testis tubule cultures labeled with 32PO4. Histones were analyzed by two systems of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography of the gels. Of the 32PO4 detected in histones, 95% was incorporated by certain modified forms of the H2A variants H2A.1 and H2A.X. Phosphorylation sites were mapped to N- and C-terminal regions of the modified variants by SDS gel electrophoresis and autoradiography of peptides generated by cleavage of in vitro-labeled proteins with N-bromosuccinimide. Incorporation rates differed for N- and C-terminal regions from different modified forms, demonstrating a complex pattern of H2A phosphorylation in the mouse testis.     

Novel leukocyte agonists are released by endothelial cells exposed to peroxide.

Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.     

Substantial quantities of the high energy derivative oligophosphoglyceroyl-ATP are located in mitochondria in rat liver

Perfusion with [8-14C]adenosine demonstrated the likely existence in rat liver of oligophosphoglyceroyl-ATP (OPG-ATP). Purification followed by assay with a new specific 3' phosphodiesterase confirmed this. The quantities present were 5-10-fold those found previously and comparable to total soluble nucleotides. OPG-ATP was also purified from the mitochondrial fraction, shown to co-distribute with succinate dehydrogenase and can be co-purified with an enzyme confined to intermembrane space.     

Purification of the arom multienzyme aggregate from Euglena gracilis.

The arom multienzyme complex that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway has been purified up to 2000-fold from Euglena gracilis. The native arom aggregate has a molecular weight of approx. 249 000 based on a sedimentation coefficient of 9.5 and Stokes radius of 60 angstrom. A comparison between the arom aggregates of Neurospora crassa and Euglena gracilis and the possible phylogenetic relationships between the organisms are discussed.