The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNA Gln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNA Gln through the transamidation of misacylated Glu-tRNA Gln , functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNA Gln in B. subtilis and that glutamyl-tRNA Gln amidotransferase is a novel and essential component of the translational apparatus.

The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastasedeficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1β gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastasedeficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60-80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40-to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.is the prototypic member of the serpin superfamily and one of the most abundant serine protease inhibitors in the circulation (1). As an acute-phase protein, AAT is thought to play an important role in limiting host-tissue injury triggered by proteases, particularly neutrophil elastase (NE). The clinical relevance of AAT is highlighted in individuals with inherited deficiency in circulating AAT, who have increased susceptibility to early-onset pulmonary emphysema, liver and pancreatic diseases, and in rare cases to panniculitis and vasculitis (2). It has been assumed that in AAT-deficiency the protease/antiprotease balance is shifted toward NE, which leads to extensive tissue damage, particularly in causing emphysema. Therefore, augmentation of circulating AAT was introduced 25 y ago to treat emphysema patients with severe PiZZ (Glu342Lys) AAT deficiency (3). Because clinical trials of AAT augmentation therapy use historical data as controls, the benefit of AAT therapy for PiZZ patients remains under debate (4-6), although in most cases therapy offers disease stabilization. The uncertainty surrounding the efficacy of augmentation therapy also reflects the incomplete understanding of the properties of the AAT protein, which can be affected by the isolation methods from plasma.Although the a...

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNAGIn in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

Inflammatory cytokines mediate inflammatory bowel diseases (IBDs) and cytokine blocking therapies often ameliorate the disease severity. IL-32 affects inflammation by increasing the production of IL-1, TNFα, and several chemokines. Here, we investigated the role of IL-32 in intestinal inflammation by generating a transgenic (TG) mouse expressing human IL-32γ (IL-32γ TG). Although IL-32γ TG mice are healthy, constitutive serum and colonic tissue levels of TNFα are elevated. Compared with wild-type (WT) mice, IL-32γ TG mice exhibited a modestly exacerbated acute inflammation early following the initiation of dextran sodium sulfate (DSS)-induced colitis. However, after 6 d, there was less colonic inflammation, reduced tissue loss, and improved survival rate compared with WT mice. Associated with attenuated tissue damage, colonic levels of TNFα and IL-6 were significantly reduced in the IL-32γ TG mice whereas IL-10 was elevated. Cultured colon explants from IL-32γ TG mice secreted higher levels of IL-10 compared with WT mice and lower levels of TNFα and IL-6. Constitutive levels of IL-32γ itself in colonic tissues were significantly lower following DSS colitis. Although the highest level of serum IL-32γ occurred on day 3 of colitis, IL-32 was below constitutive levels on day 9. The ability of IL-32γ to increase constitutive IL-10 likely reduces TNFα, IL-6, and IL-32 itself accounting for less inflammation. In humans with ulcerative colitis (UC), serum IL-32 is elevated and colonic biopsies contain IL-32 in inflamed tissues but not in uninvolved tissues. Thus IL-32γ emerges as an example of how innate inflammation worsens as well as protects intestinal integrity.

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The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC).

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Background:The maturation process of IL-33 (IL-1F11) remains unclear. Results: IL-33 ligand affinity column isolates neutrophil proteinase 3. Conclusion: PR3 is an IL-33-processing enzyme. Significance: PR3 has a dual function in IL-33 biological activity.