5-Methylthioribose kinase. A new enzyme involved in the formation of methionine from 5-methylthioribose.

The presence of a previously unidentified enzyme, tentatively designated 5-methylthioribose kinase, has been demonstrated in cell-free extracts of Enterobacter aerogenes. The enzyme catalyzes the ATP-dependent phosphorylation of 5-methylthioribose. ADP is one of the products of the reaction and, based on functional group analyses, the other product is 5-methylthioribose 1-phosphate. A 40-fold purified enzyme preparation has been obtained from a cell-free extract of E. aerogenes. Activity of the partially purified enzyme is totally dependent on the presence of a divalent cation and a sulfhydryl reagent. The substrate specificity of the enzyme is quite narrow, and the Km values for ATP and 5-methylthioribose are 7.4 X 10(-5) M and 8.1 X 10(-6) M, respectively. These results suggest that 5-methylthioribose kinase may be a primary enzyme involved in the recycling of the methylthio group of 5-methylthioribose back into methionine.     

Covalent binding of proteinases in their reaction with alpha 2-macroglobulin.

Although it is known that most of the plasma proteinase inhibitors form complexes with proteinases that are not dissociated by SDS (sodium dodecyl sulphate), there has been disagreement as to whether this is true for alpha 2M (alpha 2-macroglobulin). We have examined the stability to SDS with reduction of complexes between alpha 2M and several 125I-labelled proteinases (trypsin, plasmin, leucocyte elastase, pancreatic elastase and papain) by gel electrophoresis. For each enzyme, some molecules were separated from the denatured alpha 2M chains, but amounts ranging from 8.3% (papain) to 61.2% (trypsin) were bound with a stability indicative of a covalent link. Proteolytic activity was essential for the covalent binding to occur, and the proteinase molecules became attached to the larger of the two proteolytic derivatives (apparent mol.wt. 111 000) of the alpha 2M subunit. We take this to mean that cleavage of the proteinase-susceptible site sometimes leads to covalent-bond formation between alpha 2M and proteinase. Whatever the nature of this bond, it does not involve the active site of the proteinase, as bound serine-proteinase molecules retain the ability to react with the active-site-directed reagent [3H]Dip-F (di-isopropyl phosphorofluoridate). Our conclusion is that the ability to form covalent links is not essential for the inhibitory capacity of alpha 2M. It may, however, help to stabilize the complexes against dissociation or proteolysis.     

Cysteine auxotrophs of Salmonella typhimurium which grow without cysteine in a hydrogen/carbon dioxide atmosphere.

Cysteine auxotrophs of Salmonella typhimurium mutated in cysB, cysI or cysJ grew with sulphate as a sulphur source when incubated under a hydrogen/carbon dioxide atmosphere. Yields obtained under these conditions were equivalent to those characteristic of wild-type S. typhimurium. The same mutants failed to grow with sulphate as a sulphur source when incubated aerobically. Auxotrophs mutated in cysA, cysC, cysD, cysE, cysG and cysH required cysteine for growth under both incubation conditions. The results suggest that mutations in cysB (regulation of the several cys operons) and also cysI and cysJ (sulphite reductase activity) can be circumvented during anaerobic growth under hydrogen.     

Resistance of Cysts of Amoebae to Microbial Decomposition

Cyst walls of Acanthamoeba rhysodes, A. palestinensis, A. castellanii, and one other strain of Acanthamoeba contain 36 to 45% protein and 20 to 34% carbohydrate. More than half of the protein in the walls of A. palestinensis, A. castellanii and Acanthamoeba sp. is accessible to and hydrolyzed by protease, and 67 to 69% of the carbohydrate of A. palestinensis and A. rhysodes walls is hydrolyzed by cellulase. The extent of hydrolysis of walls of the other amoebae by these enzymes is appreciably less, and chitinase and β-1,3-glucanase have no detectable effect. Protease solubilizes 10% or less of the weight of intact cysts, and no solubilization is observed with cellulase. Walls of A. palestinensis are extensively degraded in soil, the activity is less with A. rhysodes, and little attack on the other amoebae occurs. When added to soil, the protozoa excyst and grow for short periods, the trophozoites then die, and chiefly cysts persist thereafter.     

Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

Summary An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm² in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, β-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlied-atmosphere chamber which was gassed with 95% O₂ and 5% CO₂. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30°C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, aflatoxin B₁, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.     

Superinfection exclusion by bacteriophage T7.

Only two of the early genes of bacteriophage T7 were found to play a significant role in exclusion of superinfecting bacteriophage T3 particles; genes 0.3 and 1. Protein synthesis by the preinfecting phage particle was not required for efficient exclusion. These findings are discussed with regard to the known functions of these genes during T7 development.     

Integrin-linked kinase tunes cell–cell and cell-matrix adhesions to regulate the switch between apoptosis and EMT downstream of TGFβ1

Epithelial-mesenchymal transition (EMT) is a morphogenetic process that endows epithelial cells with migratory and invasive potential. Mechanical and chemical signals from the tumor microenvironment can activate the EMT program, thereby permitting cancer cells to invade the surrounding stroma and disseminate to distant organs. Transforming growth factor β1 (TGFβ1) is a potent inducer of EMT that can also induce apoptosis depending on the microenvironmental context. In particular, stiff microenvironments promote EMT while softer ones promote apoptosis. Here, we investigated the molecular signaling downstream of matrix stiffness that regulates the phenotypic switch in response to TGFβ1 and uncovered a critical role for integrin-linked kinase (ILK). Specifically, depleting ILK from mammary epithelial cells precludes their ability to sense the stiffness of their microenvironment. In response to treatment with TGFβ1, ILK-depleted cells undergo apoptosis on both soft and stiff substrata. We found that knockdown of ILK decreases focal adhesions and increases cell–cell adhesions, thus shifting the balance from cell–matrix to cell–cell adhesion. High cell–matrix adhesion promotes EMT whereas high cell–cell adhesion promotes apoptosis downstream of TGFβ1. These results highlight an important role for ILK in controlling cell phenotype by regulating adhesive connections to the local microenvironment.