Regulation of the hapten-specific T cell response. I. Preferential induction of hyporesponsiveness to the D-end of the major histocompatibility complex in the hapten-specific cytotoxic T cell response

In this paper we have examined the phenomenon of hapten-specific tolerance in the cytolytic T lymphocyte (CTL), using the trinitrophenyl (TNP) and azobenzenearsonate haptens. We found that the H-2 K and H-2 D-end restricted CTL in H-2a mice are differentiable in the ease with which they are tolerized to the TNP hapten. With TNP modified syngeneic spleen cells (TNP-SC), or low amounts of trinitrobenzylsulfonic acid as tolerogen, preferential hyporesponsiveness of D-end restricted CTL can be observed. Larger doses of hapten, e.g. a higher amount of trinitrobenzylsulfonic acid, will tolerize both K- and D-end restricted TNP-specific CTL in H-2a mice. The phenomenon of preferential D-end restricted CTL hyporesponsiveness is not observed in H-2d, H-2k, or H-2b mice, nor is it observed in H-2a mice with respect to the azobenzenearsonate hapten. We have also shown that the clones of CTL responsible for lysis of TNP-modified allogeneic targets (cross-reactive lysis) in H-2a mice probably overlap with the D-end restricted TNP-specific CTL since D-end restricted hyporesponsiveness induced by intravenous injection of TNP spleen cells also results in the elimination of cross-reactive lysis of TNP-modified allogeneic targets. The possible mechanisms of preferential D-end hyporesponsiveness to the TNP hapten in the H-2a mice as well as its significance and relationship to previous work in this area are discussed in this paper.     

Activation of ppGpp-3'-pyrophosphohydrolase by a supernatant factor and ATP

The breakdown of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) into GDP and PPi is catalyzed by a Mn2+-dependent 3'-pyrophosphohydrolase, the translation product of the spoT gene. The escherichia coli enzyme is normally found to be associated with the "crude" ribosome fraction. It is reported here that the guanosine 5'-diphosphate, 3'-diphosphate 3'-pyrophosphohydrolase activity in this fraction is activated by ATP in the presence of a relatively heat-stable, low molecular weight, supernatant factor (BS100). This stimulation is not due to a removal of reaction products such as by the phosphorylation of GDP to GTP or by the hydrolysis of PPi. Hydrolysis of ATP is probably required because neither adenosine 5'-(3-thio)triphosphate nor adenosine 5'-(beta, gamma-imido)triphosphate can substitute for ATP. Levallorphan, a morphine analog, which had been shown to inhibit in vivo ppGpp degradation, inhibits specifically the stimulation of ppGpp hydrolysis by ATP and the supernatant factor. The possible relationship of this system and the in vivo energy-dependent control of ppGpp degradation is discussed.     

Dipeptide inhibitors of ubiquitin-mediated protein turnover prevent growth factor-induced neurite outgrowth in rat pheochromocytoma PC12 cells.

Dipeptide inhibitors of the ubiquitin-dependent proteolysis pathway governed by N-terminal recognition (N-end rule) in reticulocyte lysates significantly suppress NGF- and bFGF-induced neurite outgrowth in rat pheochromocytoma PC12 cells, but do not cause retraction of already formed neurites. Peptides which do not inhibit proteolysis are also without effect on PC12 cell differentiation. Suppression of neurite outgrowth is readily reversible upon removal of the inhibitors. These data demonstrate a requirement for specific protein turnover in the process of neuron-like differentiation in PC12 cells and provide the first demonstration of a physiological role for the N-end rule.     

Tandem mass spectrometry data quality assessment by self-convolution

Background  

Many algorithms have been developed for deciphering the tandem mass spectrometry (MS) data sets. They can be essentially clustered into two classes. The first performs searches on theoretical mass spectrum database, while the second based itself on de novo sequencing from raw mass spectrometry data. It was noted that the quality of mass spectra affects significantly the protein identification processes in both instances. This prompted the authors to explore ways to measure the quality of MS data sets before subjecting them to the protein identification algorithms, thus allowing for more meaningful searches and increased confidence level of proteins identified.     

Modifications of membrane lipids in response to wounding of Arabidopsis thaliana leaves

Mechanical wounding of Arabidopsis thaliana leaves results in modifications of most membrane lipids within 6 hours. Here, we discuss the lipid changes, their underlying biochemistry, and possible relationships among activated pathways. New evidence is presented supporting the role of the processive galactosylating enzyme SENSITIVE TO FREEZING2 in the wounding response.     

Cloning, high-level expression, purification, and properties of a novel endo-beta-1,4-mannanase from Bacillus subtilis G1 in Pichia pastoris

A novel gene coding for an endo-beta-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The beta-mannanase showed an identity of 90.2-92.9% (< or =95%) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified beta- mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDITOF mass spectrometry. The recombinant beta-mannanase had an optimum temperature of 45 degrees C and optimum pH of 6.5. The enzyme was stable at temperatures up to 50 degrees C (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions (Hg2+, Pb2+, and Co2+) substantially inhibited the recombinant beta-mannanase. The chemical additives including detergents (Triton X- 100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the beta-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.