Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.     

P, P-bis(5′-adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl-tRNA synthetase

Bovine tryptophanyl-tRNA synthetase (TrpRS, E.C. 6.1.1.2) is unable to catalyze in vitro formation of Ap₄A in contrast to some other aminoacyl-tRNA synthetases. However, in the presence of -tryptophan, ATP-Mg²⁺ and ADP the enzyme catalyzes the Ap₃A synthesis via adenylate intermediate. Ap₃A (not Ap₄A) may serve as a substrate for TrpRS in the reaction of E·(Trp AMP) formation and in the tRNA^Trp charging. The K_m value for Ap₃A was higher than the K_m for ATP (approx. 1.00 vs. 0.22 mM) and V_max was 3 times lower than for ATP. The Zn²⁺-deficient enzyme catalyzes Ap₃A synthesis in the absence of exogenous ADP due to ATPase activity of Zn²⁺-deprived TrpRS. The inability of mammalian TrpRS to synthesize Ap₄A, might be considered as a molecular tool preventing the removal of Zn²⁺ due to chelation by Ap₄A and therefore preserving the enzyme activity.     

Polypeptide release factors in prokaryotes and eukaryotes: same function, different structure.

Although the eukaryotic (eRF1) and prokaryotic (RF2) polypeptide release (translation termination) factors are functionally similar, they turn out to be very different in overall shape and architecture and in the location of key functional elements.     

Partial purification and characterization of a novel human factor that augments the expression of class I MHC antigens on tumour cells

A cytokine which augments the expression of major histocompatibility complex (MHC) I antigens on K562 and gastric carcinoma tumour (HR) cells, has been isolated from the culture supernatant of Concanavalin-A (Con-A) activated human peripheral blood mononuclear cells. The factor, termed MHC augmenting factor (MHC- AF) has been partially purified by Sephadex G- 100 column chromatography, preparative isoelectric focusing and HPLC with ion- exchange as well as sizing columns. MHC-AF activity is associated with a 35 kDa molecule which has pI of 6.0. Interferon (IFN)-α, \, tumour necrosis factor (TNF), Interleukin (IL)-2, IL-4, IL-5 and IL-7 had no significant effect in MHC- AF bioassay, but IFN-γ had significant MHC-AF activity. Antibodies to IFN-α, IFN-\ and TNF-α did not block the activity of MHC-AF, but anti-IFN-y antibodies could partially neutralize the activity. However, unlike IFN-γ, MHC-AF activity was resistant to pH 2.0 treatment. Purified MHC-AF preparations did not have any activity in WISH cell/encephalo myocarditis virus (EMC) IFN bioassays. In addition, anti-IFN-y affinity column did not retain MHC-AF activity. These results indicate that a MHC-AF distinct from IFN-γ, is produced by activated human mononuclear cells.     

Triton channels are sensitive to divalent cations and protons

Addition of Triton X-100 to planar bilayers composed of dioleoyl phosphatidyl choline, diphytanoyl phosphatidyl choline or mono-oleoyl glycerol induces single channel-like events when electrical conductivity across the bilayer is measured. Addition of divalent cations or protons causes channels to disappear; single channel conductance of remaining channels is not significantly altered; addition of EDTA or alkali (respectively) reverses the effect. It is concluded that sensitivity to divalent cations and protons need not be dependent on specific channel proteins or pore-forming toxins, but may be a feature of any aqueous pore across a lipid milieu.We are grateful to Dr. D.T. Edmonds and Prof. R.J.P. Williams for critical discussion, to Glenn Alder for technical assistance, to Ms. B. Bashford and Ms. S.G. Pelc for preparing the paper, and to the Cell Surface Research Fund, the Royal Society (A.A.L.), UNESCO (Molecular and Cell Biology Network) and The Wellcome Trust for financial support.