A conserved domain in prokaryotic bifunctional FAD synthetases can potentially catalyze nucleotide transfer

Biosynthesis of flavin adenine dinucleotides in most prokaryotes is catalyzed by a family of bifunctional flavin adenine dinucleotide (FAD) synthetases. These enzymes carry out the dual functions of phosphorylation of flavin mononucleotide (FMN) and its subsequent adenylylation to generate FAD. Using various sequence analysis methods, a new domain has been identified in the N-terminal region that is well conserved in all the bacterial FAD synthetases. We also identify remote similarity of this domain to the nucleotidyl transferases and, hence, this domain is suggested to be invloved in the adenylylation reaction of FAD synthetases

IoT Based Automated Saline Drip and Hand Movement Detection System for Critical Care Patients

ABSTRACT: The major goal of this project is to develop a saline drip detection and control system used to inject saline or intravenous fluids into a patient's veins. This project presents an IoT-based system to address issues such as fluid backflow, blood loss, and so on. Solution to concerns like fluid backflow and blood loss, among others. As a result, we devised the Automated Intravenous Drip Monitoring System. In this project, we utilize an Arduino-based microcontroller to manage the drop rate, detect tube obstruction, and warn the user when the drips are emptied. Here we are going to monitor the drip rate whether it is low or over, by using ultrasonic sensor. By using the MEMS motion sensor, we can also detect the patient's movement while taking drips after they have undergone some surgery or in ICU condition. In addition, we are going to estimate the possibilities of blood clotting in the veins of the patient. If motion is detected in the infusion hose, the sensors will alert the doctors or nurses with a buzzer and an LED light. The flow rate and infusion interruption problems were monitored by transmitting the data wirelessly via Bluetooth via their smart phones using mobile applications and computer-based applications. As a result, at the nurse's station, the developing system can closely monitor the drip infusion situation of numerous patients.</jats:p

Increased Levels of the Heat Shock Protein (hsp) 70 Contribute as a Pro-Survival and Pro-Growth Mechanism in the Transformation of Bone Marrow Progenitor Cells.

Abstract Hsp70 is an ATP-dependent molecular chaperone that assists in the folding of native proteins into active conformation and prevents aggregation of misfolded and mutated abnormal proteins. In normal non-transformed cells, the expression of hsp70 is low and largely stress-inducible due to misfolded and denatured proteins. Recent studies in our laboratory have demonstrated that human acute leukemia cells abundantly express hsp70, which exerts strong antiapoptotic effects upstream and downstream of the mitochondria. Additionally, as compared to the controls, the mouse myeloid 32D or BaF3 cells transformed by Bcr-Abl or FLT-3 also show increased expression of hsp70. To further elucidate the pro-survival and pro-growth effects of hsp70 and its role in the leukemia transformation, we created stable hsp70 transfectants of the IL-3-dependent 32D and BaF3 (normally maintained in culture in IL-3 containing 10% WEHI medium), i.e., 32D/hsp70 and BaF3/hsp70 cells. These cells displayed 3 to 5 fold higher levels of hsp70, as compared to the control 32D or BaF3 cells. Both 32D/hsp70 and BaF3/hsp70 cells showed significantly improved growth and survival supported by 10% WEHI medium. Following culture in 0%, and less so in 1%, WEHI medium for 24 hours, 32D and BaF3 cells undergo cell cycle G1 phase accumulation, with corresponding decline in the % of cells in the S phase. Following this exposure, they also show markedly increased apoptosis and loss of clonogenic survival, as determined by the colony growth assays in methylcellulose. In contrast, under similar conditions of exposure to reduced % of WEHI conditioned medium, 32D/hsp70 and BaF3/hsp70 cells displayed significantly less accumulation in G1 phase, as well as reduced loss of clonogenic survival and apoptosis (p&amp;lt;0.05). This was also associated with reduced loss of mitochondrial membrane potential and increased accumulation of reactive oxygen species. Notably, following IL-3 withdrawal, exposure to 10 ng/ml of G-CSF for 72 hours induced significantly less differentiation of 32D/hsp70 versus 32D cells, as determined by increase in the % of cells expressing CD11b and GR1 (determined by specific antibody staining and flow cytometry) or by evaluation of the morphologic features of differentiation (p&amp;lt;0.05). Western blot analyses demonstrated that both 32D/hsp70 and BaF3/hsp70 cells, compared to their controls, possessed significantly higher expression of IL-3β receptor (R) and pSTAT5. Importantly, the supernatants of hsp70 overexpressing cells, compared to their controls, also showed higher levels of IL-3, as detected by an ELISA. In addition, BaF3/hsp70, versus the control cells, showed increased DNA binding activity and transactivation by the AP1 transcription factor, utilizing a protein/DNA binding array assay (Panomics, Redwood City, CA) and AP-1-luciferase cis-reporting analysis (Stratagene, La Jolla, CA), respectively. These findings strongly suggest that increased hsp70 levels confer a growth and survival advantage through an IL3- IL-3βR-STAT5-dependent mechanism in the marrow progenitor cells, which may contribute to the transformation induced by leukemia associated oncoproteins.</jats:p

Histone Deacetylase Inhibitors LBH589 and LAQ824 Deplete Ezh2 and Associated Polycomb Repressive Complex 2/3 Proteins Resulting in Downregulation of HOXA9 and MEIS1 Expression in Human Acute Leukemia Cells.

Abstract Human enhancer of Zeste homolog (Ezh2) protein belongs to Polycomb Repressive Complex (PRC) 2, which also includes Eed and Suz12. Ezh2 has been shown to promote cellular transformation, and increased Ezh2 expression has been strongly correlated with the invasiveness of prostate and breast cancers. The enzymatically-active Ezh2-containing, PRC2 complex possesses histone methyl transferase (HMTase) activity mediated by the SET domain of Ezh2, which is involved in the methylation of histone (H) 3, lysine (K)-27 and −9. Through this, the PRC2 complex regulates the expression of homeobox domain containing HOX family of transcription factors including HOXA9 and MEIS1, which have been shown to be involved in human leukemogenesis. Co-expression of HOXA9 and MEIS1 is common in acute myeloid leukemia and collaborates in the leukemogenesis. In the present studies, we determined the effect of hydroxamate histone deacetylase inhibitors LBH589 and LAQ824 on Ezh2 and PRC2 complex proteins and their activity in the cultured (K562, LAMA-84, U937 and HL-60) and primary human AML cells. Exposure to 10 to 100 nM of LBH589 or LAQ824 for 24 hours, in a dose dependent manner depleted the protein levels of Ezh2, as well as reduced Suz12 and Eed levels in the cultured and primary leukemia cells. This was associated with decreased levels of the tri- and dimethylated K27 and increased acetylation of K27, both on H3. Correspondingly, these H3 modifications were associated with a significant decline in the levels of HOXA9 and MEIS1. As has been previously reported, treatment with LBH589 and LAQ824 induced p21, as well as caused cell cycle growth arrest in the G1 phase and apoptosis of the cultured and primary AML cells. We next determined whether knockdown of Ezh2 by siRNA to Ezh2 also induces growth inhibitory and cytotoxic effects against the leukemia cells. In the cultured and primary acute leukemia cells, knockdown of Ezh2 expression (by ~80%) by Ezh2 siRNA, but not by the control siRNA, was associated with depletion of Suz12 levels and increased H3K27 acetylation. This was associated with increase in the % of cells in the G1 phase of the cell cycle and significant inhibition of their clonogenic survival in colony growth assays. Co-treatment with LBH589 and siRNA to Ezh2 caused further decline in the Ezh2 levels and increased loss of clonogenic survival of the leukemia cells. These findings suggest that down modulation of Ezh2 and PRC2 complex and its HMTase activity may inhibit clonogenic survival of human acute myeloid leukemia cells. Additionally, combined effect of the knockdown of Ezh2 and its activity along with treatment with LBH589 or LAQ824 may have an improved anti-leukemia efficacy, especially against human AML where HOXA9 and MEIS1 genes are deregulated.</jats:p

Co-Treatment with a Novel Heat Shock Protein (hsp) 90 Inhibitor IPI504 and the Histone Deacetylase Inhibitor (HDI) Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA): A Highly Active Combination Against Wild Type or Mutant Bcr-Abl-T315I or FLT-3-ITD-Containing Human Leukemia Cells.

Abstract Hsp90 is an ATP-dependent molecular chaperone, which helps in folding its client proteins, e.g., Bcr-Abl, FLT-3, c-Raf and Akt, into active conformation. Geldanamycin analogue, 17-AAG (Kosan Biosciences Inc., Hayward, CA) inhibits the chaperone function of hsp90, which promotes polyubiquitylation and proteasomal degradation of the misfolded hsp90 client proteins. We recently reported that, by inhibiting the activity of histone deacetylase 6, the hydroxamate HDIs such as vorinostat (Merck &amp; Co., Inc.) induce acetylation and inhibition of hsp90, thus also causing the depletion of its client proteins. In the present studies, we determined the anti-leukemia effects of the novel, highly soluble, hsp90 antagonist IPI504 (Infinity Pharmaceuticals), which, in vitro and in vivo, interconverts with 17-AAG, ± vorinostat, against human cultured or primary, wild type or mutant Bcr-Abl or mutant FLT-3 containing acute leukemia cells. Treatment with IPI504 (0.5 to 2.0 μM) for 24 to 48 hours, in a dose dependent manner, induced apoptosis of WT Bcr-Abl-expressing K562 and LAMA-84 cells. This was associated with attenuation of the levels of Bcr-Abl, pCrkL, pSTAT5, c-Raf and pAkt. In a dose dependent manner (50 to 500 nM for 48 hours), IPI504 also induced apoptosis of FLT-3 internal tandem duplication (ITD)-containing human acute leukemia MV4-11 cells, which was associated with attenuation of the levels of FLT-3, pAkt, pSTAT5, pERK1/2. Notably, treatment with IPI504 induced similar level of apoptosis of mouse bone marrow BaF3 cells, which had been transformed and rendered IL-3 independent for growth by ectopic expression of WT Bcr-Abl, its P-loop (Bcr-Abl-E255K) or highly imatinib mesylate (IM) resistant, contact-inhibition (Bcr-Abl-T315I) point mutant. This was also associated with attenuation of the levels of WT and mutant Bcr-Abl-E255K or Bcr-Abl-T315I. In previous studies we had demonstrated that treatment with vorinostat depletes WT and mutant Bcr-Abl levels and induces apoptosis of expressing human leukemia cells. Therefore, we determined the effect of the co-treatment of IPI504 (1.0 μM) and vorinostat (1.0 μM) against cultured or primary human CML cells. Co-treatment with IPI504 and vorinostat induced significantly more apoptosis of K562 and MV4-11 cells, which was associated with more depletion of WT-Bcr-Abl and FLT-3-ITD levels in K562 and MV4-11 cells, respectively. Notably, co-treatment with IPI504 and vorinostat, versus treatment with either agent alone, also induced more apoptosis of primary CML cells (4 samples) derived from patients with IM-resistant CML, including a sample of cells documented to have Bcr-Abl-T315I mutation. Additionally, as compared to treatment with either agent alone, the combination of IPI504 and vorinostat also induced more apoptosis of primary AML cells (4 samples), including two samples that contained FLT-3-ITD. These findings demonstrate that the combination of IPI504 with vorinostat exerts a high level of in vitro activity against FLT-3-ITD-containing acute leukemia, as well as against highly IM-resistant mutant Bcr-Abl-expressing leukemia cells.</jats:p

Co-Treatment with the hsp90 Inhibitor 17-Dimethylaminoethylamino-17-Demethoxygeldanamycin (DMAG) and Histone Deacetylase Inhibitor (HDI) Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA): A Highly Active Combination Against Human Mantle Cell Lymphoma (MCL) Cells.

Abstract We have previously reported that agents that inhibit ATP binding and chaperone function of hsp90 are highly active against wild type and mutant Bcr-Abl and mutant FLT-3 containing human acute leukemia cells. In the present studies, we determined the effects of a more soluble and potent geldanamycin analogue, DMAG (Kosan Biosciences Inc.), and/or hydroxamate histone deacetylase inhibitor (HHDI), vorinostat (Merck &amp; Co., Inc.), against human MCL Jeko1 and MO2058 cells. These cells contain the characteristic MCL-associated chromosomal translocation t(11; 14)(q13;q32), which results in the overexpression of cyclin D1. Recently, HHDIs, such as vorinostat, have been shown to inhibit HDAC6, which results in the acetylation of hsp90 and inhibition of its ATP binding and chaperone function. Treatment with vorinostat (0.5 to 2.0 μM) induced the accumulation of the cells in the G1 and DMAG (0.1 to 0.5 μM) in the G2/M phase of the cell cycle. Both agents induced apoptosis in a dose-dependent manner (up to 50%). While vorinostat induced both p21 and p27 levels, DMAG only increased the intracellular levels of p21. Treatment with either agent depleted the intracellular levels of c-Myc, c-Raf, Akt and cdk4 in a dose dependent manner. It is well established that the chaperone association with hsp90 maintains Akt, c-Raf, cyclin D1 and cdk4 in the native and active conformation, and inhibition of hsp90 promotes their polyubiquitylation and proteasomal degradation. Notably, co-treatment with DMAG (e.g., 0.25 μM) and vorinostat (e.g., 2.0 μM), more than either agent alone, markedly attenuated the levels of cyclin D1 and cdk4, as well as the levels of c-Myc, c-Raf and Akt. The combination of DMAG and vorinostat also induced significantly more apoptosis of Jeko1 and MO2058 cells, as compared to the treatment with either agent alone (p &amp;lt; 0.01). These findings demonstrate that the combined treatment with vorinostat and DMAG is highly active against human MCL cells, and support the rationale to determine the in vivo efficacy and safety of the combination against human MCL.</jats:p

Treatment with the Heat Shock Protein (hsp) 90 Inhibitor17-Dimethylaminoethylamino-17-Demethoxygeldanamycin (DMAG) Abrogates the Levels and Activity of the Receptor Tyrosine Kinase TrkA in Acute Leukemia Cells.

Abstract Nerve growth factor (NGF) mediates the phosphorylation and signaling through the receptor tyrosine kinase TrkA, which has been shown to be expressed and active in the early hemopoietic progenitor cells, as well as in the K562 and TF1 leukemia cell lines and AML-ETO-expressing human acute leukemia cells. In AML, a 75-amino acid deletion mutant of TrkA (ΔTrkA) has also been demonstrated to be constitutively active as a pro-growth and pro-survival protein through ERK1/2 and Akt activation. We have previously reported that that the ATP bound molecular chaperone hsp90 binds the leukemia associated Bcr-Abl and FLT-3 tyrosine kinases as client proteins, maintaining them in a properly folded and active conformation, and that geldanamycin analogue hsp90 inhibitors disrupt this chaperone association, resulting in polyubiquitylation and proteasomal degradation of the client proteins. In the present studies, we investigated a) whether TrkA is a client protein of hsp90 and b) the effect of the novel and highly soluble hsp90 inhibitor DMAG (Kosan Biosciences Inc.) on TrkA levels and activity in mouse myeloid 32D cells with or without the ectopic expression of ΔTrkA (32D/ΔTrkA cells), as well as on endogenous levels of wild-type (WT) TrkA in K562 and TF1 cells. Exposure to 0.25 or 1.0 μM DMAG attenuated the levels of WT TrkA in K562, TF1 and 32D, as well as ΔTrkA in 32D/ΔTrkA cells. Co-treatment with the proteasome inhibitor bortezomib (100 nM) restored DMAG mediated depletion of WT TrkA in K562 cells, suggesting that DMAG induced the polyubiquitylation and degradation of TrkA by the 26S proteasome. In K562 cells, immunoprecipitation (IP) with monoclonal anti-TrkA antibody followed by immunoblot (IB) analyses with anti-hsp90 antibody (or IP with anti-hsp90 followed by IB with anti-TrkA antibody) showed that TrkA binds to hsp90, which is inhibited by treatment with DMAG. Following suspension of K562 cells in a serum free medium containing 100 ng/ml of NGF, the levels of pTrkA, pERK1/2 and pAkt significantly increased within 5 to 10 minutes. Co-treatment with 1.0 μM DMAG inhibited pTrkA and pERK1/2 induction, suggesting that hsp90 chaperone function may be required for TrkA activity. Exposure to DMAG also depleted the levels of the other hsp90 client proteins, including c-Raf, Akt and Bcr-Abl in K562 cells, which was associated with growth arrest and apoptosis in a dose-dependent manner. These findings demonstrate that TrkA may be an hsp90 client protein, and hsp90 inhibition by treatment with DMAG would deplete WT or mutant TrkA levels and activity in human leukemia cells. These findings suggest that hsp90 inhibitors may be effective against human acute leukemia cells that may depend on the activity of mutant or WT TrkA for growth and survival.</jats:p