Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression

The Mixed-Lineage Leukemia gene (MLL/HRX/ALL1) encodes a large nuclear protein homologous to Drosophila trithorax that is required for the maintenance of HOX gene expression. MLL is cleaved at two conserved sites generating N320 and C180 fragments, which heterodimerize to stabilize the complex and confer its subnuclear destination. Here, we purify and clone the protease responsible for cleaving MLL. We entitle it Taspase1 as it initiates a class of endopeptidases that utilize an N-terminal threonine as the active site nucleophile to proteolyze polypeptide substrates following aspartate. Taspase1 proenzyme is intramolecularly proteolyzed generating an active 28 kDa alpha/22 kDa beta heterodimer. RNAi-mediated knockdown of Taspase1 results in the appearance of unprocessed MLL and the loss of proper HOX gene expression. Taspase1 coevolved with MLL/trithorax as Arthropoda and Chordata emerged from Metazoa suggesting that Taspase1 originated to regulate complex segmental body plans in higher organisms.     

IRAK contributes to burn-triggered myocardial contractile dysfunction

Major burn injury causes myocardial contractile dysfunction, but the molecular basis of this physiological response is incompletely understood. Previous studies demonstrated a role for the interleukin-1 receptor-associated kinase (IRAK) in the cardiac response to acute lipopolysaccharide administration as well as congestive heart failure. In this study, we examined the contribution of IRAK to burn-mediated cardiac responses. After burn injury, hearts from wild-type and IRAK-deficient mice were compared for intracellular signaling pathway activation and contractile function. IRAK-deficient hearts showed impaired activation of kinases that function downstream of IRAK and were partially protected against burn-induced contractile dysfunction. The findings demonstrate that IRAK and the Toll/interleukin-1 pathways participate in the response to large body surface area burns that leads to impaired cardiac contractility.     

Homocysteine altered ROS generation and NO accumulation in endothelial cells

Mild hyperhomocysteinemia (HHcy) is a risk factor for vascular disease and is closely associated with endothelial dysfunction. Oxidative stress and decreased nitric oxide (NO) bioavailability were reported in HHcy-induced vascular injury; however, the exact relationship is not understood. We thus directly determine the production of reactive oxygen species (ROS) and NO in cultured endothelial cells (HUVECs) to demonstrate the correlated variation between ROS and NO induced by Hcy (homocysteine), Cys (cysteine), another thiol compound, and Met (methionine), precursor of HHcy in animal study. HUVECs were treated with Hcy, Cys, or Met for 0.5 or 22-24 h; ROS generation was detected by DCF fluorescence with flow cytometry and NO by chemiluminescence. In non-cytotoxic (<1.0 mM) concentration ranges, Met exerted no effects on either ROS production or NO concentration, Cys decreased ROS production and increased NO in both short-term (0.5 h) and long-term (22-24 h) treatments; Hcy, however, induced a biphasic effect on ROS production, i.e., inhibitory at 0.5 h but stimulatory at 24 h. The maximal stimulation by Hcy (0.25 mM) was significantly reduced by co-incubation (12 h) with estrogen (1 microM). Hcy caused an early (0.5 h) increase of medium NO which was absent in long-term Hcy treatment. The oxidative stress caused by long-term Hcy incubation could be ameliorated by estrogen, consistent with earlier in vivo observations that estrogen prevents HHcy-induced injury.     

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR

We have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis, using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granuloma-specific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis, as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo. The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.     

Morphological and chemical changes in the attached cells of Pseudomonas aeruginosa as primary biofilms develop on aluminium and CaF2 plates

AIMS: To investigate the morphological and chemical changes in attached cells of Pseudomonas aeruginosa (ATCC 14886) at different stages of biofilm development on two different types of substrata. METHODS AND RESULTS: The development of primary biofilm on aluminium plates representing metals and on CaF(2) discs representing dielectric materials was monitored by FTIR microscopy, ESEM, EDAX and protein analysis by SDS-PAGE. A unique cellular feature similar in morphology to pili was observed on the surface of P. aeruginosa adhering on aluminium but not on CaF(2). Results derived from FTIR analysis confirm on both substrata the successive importance of polysaccharides and proteins during the biofilm development. These results also revealed that the increase of the ratio of carboxylates to amide I was higher with the aluminium plates than with the CaF(2) discs. The number of cells adhered and the amount of oxygen incorporated in adhered cells on the latter materials were, respectively, less and almost nil in comparison with the former. Protein analysis of the lysates of cells by SDS-PAGE revealed that expression of one protein with a molecular weight of 45 kDa, was greatly enhanced in attached cells on both substrata. However, expression of another protein with molecular weight of 35 kDa was up-regulated only in cells adhering on CaF(2) but not in those on aluminium. CONCLUSION: Depending on the nature of the surface, new proteinaceous complexes and cellular features were formed in the attachment process of P. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The pattern of P. aeruginosa cells adhering onto CaF(2) discs and aluminium plates is different. Formation of biofilm is more difficult on CaF(2) than on aluminium.     

An in vitro assessment of several enzymes for the supplementation of rabbit diets

In order to evaluate the effectiveness of several enzymes for use as feed supplements, the stabilities of these enzymes in relation to rabbit gastrointestinal content and feed processing conditions were investigated. Results showed that in glycine-HCl buffer pH 3.2, cellulase, papain and bromelain were acid tolerant while bacillus -amylase and protease were acid labile. When the buffer pH was 2.0, cellulase and papain still showed some pH resistance while bromelain was completely inactivated. With pepsin added to these buffer systems, similar results were obtained. In the gastric content system of pH 2.0, all the enzymes tested were unstable, but when the pH was 3.2 cellulase was fairly stable. Therefore, some factors in the gastric content other than pepsin might inactivate enzymes such as papain and bromelain. These factors might be the components of mineral premix in feed. On the other hand, preincubation of the enzymes with rabbit intestinal content would not affect the activities of these enzymes significantly. For the thermal stability study, it was found that, except for papain, thermal stabilities of all the enzymes tested could be significantly enhanced by mixing with the feed. Since mineral premix has an inhibitory effect on both bromelain and papain, the enzyme stabilization effect from feed could be caused by components other than mineral premix. Therefore, factors affecting the stabilities of each enzyme vary significantly and care must be taken when using these enzymes as feed supplements.     

Preclinical anti-arthritic study and pharmacokinetic properties of a potent histone deacetylase inhibitor MPT0G009

The pathology of rheumatoid arthritis includes synoviocyte proliferation and inflammatory mediator expression, which may result from dysregulated epigenetic control by histone deacetylase (HDAC). Thus, HDAC inhibitors may be useful for treating inflammatory disease. This was a preclinical study of the HDAC inhibitor, MPT0G009. The IC₅₀ values of MPT0G009 for HDAC1, 2, 3, 6 and 8 enzymatic activities were significantly lower than those for the currently marketed HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat). In addition, MPT0G009 markedly inhibited cytokine secretion and macrophage colony-stimulating factor/receptor activator of nuclear factor kappa B ligand-induced osteoclastogenesis by macrophages (50 ng/ml each). These MPT0G009 effects on cytokine secretion and osteoclast formation were reduced by the overexpression of HDAC 1 (class I HDAC) and 6 (class II HDAC) in cells, suggesting that these effects were due to the inhibition of its activity. In an in vivo rat model, oral administration of MPT0G009 (25 mg/kg) significantly inhibited paw swelling and bone destruction. Furthermore, compared with SAHA, MPT0G009 exhibited longer half-life (9.53 h for oral administration) and higher oral bioavailability (13%) in rats. These results established the preclinical anti-arthritic efficacy and pharmacokinetic parameters of MPT0G009, which may provide a new therapeutic approach for treating inflammatory arthritis.