Targeting age‐specific changes in CD4+ T cell metabolism ameliorates alloimmune responses and prolongs graft survival

Age impacts alloimmunity. Effects of aging on T‐cell metabolism and the potential to interfere with immunosuppressants have not been explored yet. Here, we dissected metabolic pathways of CD4⁺ and CD8⁺ T cells in aging and offer novel immunosuppressive targets. Upon activation, CD4⁺ T cells from old mice failed to exhibit adequate metabolic reprogramming resulting into compromised metabolic pathways, including oxidative phosphorylation (OXPHOS) and glycolysis. Comparable results were also observed in elderly human patients. Although glutaminolysis remained the dominant and age‐independent source of mitochondria for activated CD4⁺ T cells, old but not young CD4⁺ T cells relied heavily on glutaminolysis. Treating young and old murine and human CD4⁺ T cells with 6‐diazo‐5‐oxo‐l‐norleucine (DON), a glutaminolysis inhibitor resulted in significantly reduced IFN‐γ production and compromised proliferative capacities specifically of old CD4⁺ T cells. Of translational relevance, old and young mice that had been transplanted with fully mismatched skin grafts and treated with DON demonstrated dampened Th1‐ and Th17‐driven alloimmune responses. Moreover, DON diminished cytokine production and proliferation of old CD4⁺ T cells in vivo leading to a significantly prolonged allograft survival specifically in old recipients. Graft prolongation in young animals, in contrast, was only achieved when DON was applied in combination with an inhibition of glycolysis (2‐deoxy‐d‐glucose, 2‐DG) and OXPHOS (metformin), two alternative metabolic pathways. Notably, metabolic treatment had not been linked to toxicities. Remarkably, immunosuppressive capacities of DON were specific to CD4⁺ T cells as adoptively transferred young CD4⁺ T cells prevented immunosuppressive capacities of DON on allograft survival in old recipients. Depletion of CD8⁺ T cells did not alter transplant outcomes in either young or old recipients. Taken together, our data introduce an age‐specific metabolic reprogramming of CD4⁺ T cells. Targeting those pathways offers novel and age‐specific approaches for immunosuppression.     

Morphological mutation of Lentinula edodes mycelium, particularly detectable in the dikaryotic state

A morphological mutation particularly detectable in the dikaryotic state was found in Lentinula edodes. The mutant dikaryon was readily distinguishable from the normal dikaryon by the irregularly branched short hyphae, very slow hyphal growth, and sparse aerial hyphae. Genetic analysis revealed that expression of this mutation was controlled by a single recessive gene, mor-13. Linkage analysis showed that the mor-13 was not linked to either the incompatibility factors (A and B) or the five kinds of mor genes that were segregated independently of each other in a previous study. Contribution no. 380 from the Tottori Mycological Institute     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

A case of sporotrichosis caused by two genetically differentSporothrix schenckii strains

A survey for the natural occurrence of Fusarium mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN), in Dutch cereals (totaling 29 samples) harvested in 1984/1985, showed that 90%, 79% and 62% of samples were contaminated with DON, NIV and ZEN, respectively. Average contents (ng/g) in the total of positive samples were 221 (DON), 123 (NIV) and 61 (ZEN). Among the cereals examined, the highest concentrations (ng/g) was 3198 (DON), 1875 (NIV) and 677 (ZEN) in a yellow corn sample for animal feed. The results of this survey show that Dutch cereals were relatively significantly contaminated with Fusarium mycotoxins.     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

Sequence-specific DNA recognition by basic peptides.

A chiral template with C2 symmetry has been used for modeling a dimeric interface of DNA binding protein. An oligopeptide derived from the basic region of MyoD, a recently described "helix-loop-helix" class of DNA binding protein, has been tethered to the template. Among the four models which differ in chirality and polarity with respect to the arrangement of two subunits, only one dimer model with right-handed and C-terminus to C-terminus arrangement of the peptide subunits binds DNA containing native MyoD binding sequence.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex

An iron-sulfur protein has been purified from beef heart ubiquinol-cytochrome c oxidoreductase (Complex III) of the mitochondrial respiratory chain by phenyl-Sepharose column chromatography and Sephacryl S-200 gel chromatography. Depletion of most of the endogenous phospholipids in the complex was a prerequisite to the dissociation of the protein from the complex in the former chromatography. The iron-sulfur protein was nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 76 ng atoms of nonheme iron and 66 nmol of acid-labile sulfide/mg of protein. When this preparation was incubated with an iron-sulfur protein-depleted complex in the presence of soybean phospholipids, the enzymic activity was restored up to 90% of that of the parent Complex III, whereas the recovery of the activity was marginal in the absence of the phospholipids. Thus it is clear that the iron-sulfur protein is integrated into the complex with the aid of phospholipids.