Effects of targeting magnetic drug nanoparticles on human cholangiocarcinoma xenografts in nude mice

BACKGROUND:Targeting is a new therapeutic tool for malignant tumor as a result of combining nanotechnology with chemotherapeutics.The aim of our study was to investigate the effects of magnetic nanoparticles enveloping a chemotherapeutic drug on human cholangiocarcinoma xenografts in nude mice. METHODS:The human cholangiocarcinoma xenograft model was established in nude mice with the QBC939 cell line.The nude mice were randomly assigned to 7 groups. 0.9%saline or magnetic nanoparticles,including high (group 2),medium(group 4)and low(group 5)dosages, were given to nude mice through the tail vein 20 days after the QBC939 cell line was implanted.Calculations were made 35 days after treatment in order to compare the volumes,inhibition ratios and growth curves of the tumors in each group.Mice in each group were sacrificed randomly to collect tumor tissues and other organs for electron microscopy and pathological examination. RESULTS:The high and medium dosage groups were significantly different from the control group(P<0.05). The tumor inhibition ratios for the high,medium and low dosage groups were 39.6%,14.6%and 7.9%,respectively. The tumor growth curve of groups 5,4,and 2 changed slowly in turn.The high and medium groups showed cell apoptosis under an electron microscope.CONCLUSION:Magnetic nanoparticles can inhibit the growth of human cholangiocarcinoma xenografts in nude mice.     

Streptokinase—the drug of choice for thrombolytic therapy

Thrombosis, the blockage of blood vessels with clots, can lead to acute myocardial infarction and ischemic stroke, both leading causes of death. Other than surgical interventions to remove or by pass the blockage, or the generation of collateral vessels to provide a new blood supply, the only treatment available is the administration of thrombolytic agents to dissolve the blood clot. This article describes a comprehensive review of streptokinase (SK). We discuss the biochemistry and molecular biology of SK, describing the mechanism of action, structures, confirmational properties, immunogenecity, chemical modification, and cloning and expression. The production and physico-chemical properties of this SK are also discussed. In this review, considering the properties and characteristics of SK that make it the drug of choice for thrombolytic therapy.     

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome–nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP–RNC complex results in GTP-independent binding of the SRP–FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP–FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP–FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.The Escherichia coli signal recognition particle (SRP) is a complex consisting of the universally conserved protein Ffh and 4.5S RNA, which adopts a hairpin structure (1). Ffh is composed of the N-terminal domain, the G domain that harbors GTPase activity, and the C-terminal methionine-rich M domain that interacts with 4.5S RNA (2, 3) and with the signal sequence (4, 5). The N and G domains form a compact structural and functional unit termed “the NG domain.” Targeting of ribosome-nascent chain complexes (RNC) containing a signal sequence depends on the interaction of the RNC–SRP complex with the SRP receptor FtsY, which is membrane associated (6–9). FtsY and Ffh interact via their homologous NG domains and form a composite GTPase active site (10, 11). Crystal structures of the M domain reveal a hydrophobic groove used to capture signal sequences (4, 5, 12).Protein targeting is driven by highly regulated conformational rearrangements of SRP and FtsY as well as GTP hydrolysis. SRP recognizes and tightly binds to RNCs displaying a signal sequence (cargo). Next, RNC-bound SRP efficiently recruits FtsY to form a nucleotide-independent, transient early state that rearranges to a GTP-stabilized closed state (13). Ultimately, in the activated state, handover of the RNC to the Sec translocon takes place, followed by GTP hydrolysis and disassembly of the SRP–FtsY complex (14–16). These distinct conformational transitions are regulated by the ribosome and translocon in the membrane, leading to a switch from cargo recognition by SRP to cargo release (17, 18).Cryo-EM structures of bacterial SRP-bound RNCs revealed a tight cargo-recognition complex (19, 20). In the SRP–FtsY early complex an overall detachment of SRP from the ribosome was observed (21). In this state, the G domain of FtsY contacts the conserved SRP RNA tetraloop, and Ffh and FtsY interact via their N domains (21) forming a pseudosymmetric V-shaped complex positioned above the ribosomal tunnel exit. The active sites of the GTPase domains are apart from each other, explaining why the early state is inactive in GTP hydrolysis (13, 21, 22).GTP is required for SRP and FtsY to rearrange into the closed state. FRET experiments indicate that, in this state, the Ffh–FtsY NG domains adopt a conformation that resembles the intimate heterodimeric architecture observed in crystal structures (10, 11, 13). The complete SRP was crystallized in complex with the FtsY NG domain in the closed/activated state showing the NG domains docked at the distal end of the RNA hairpin (23, 24). Single-molecule total internal reflection fluorescence microscopy directly demonstrated that the Ffh–FtsY NG domains need to relocate from the tetraloop to the RNA distal end to become activated for GTP hydrolysis and to progress further in the targeting reaction (24).Although the early, closed, and activated SRP–FtsY targeting complexes have been well-characterized biochemically, the generation of distinct, conformationally homogenous closed and activated ribosome–SRP–FtsY complexes for structural studies proved to be exceedingly difficult, because the ribosome stabilizes the early state (13). We overcame this challenge by developing a robust complex preparation strategy, and describe here the cryo-EM structure of the closed state of the RNC–SRP–FtsY complex at a resolution of 5.7 Å.     

Non-antiarrhythmic drug therapy for the prevention of atrial fibrillation?

In atrial fibrillation (AF), the absence of a clear benefit of a rhythm-control strategy over a rate-control strategy seen in recent trials may be due to the fact that many of the usual antiarrhythmic strategy have significant weaknesses. Besides research efforts to improve the efficacy and safety of conventional antiarrhythmic agents, therapies directed 'upstream'of the electrical aspects of AF, towards the underlying anatomical substrate and atrial remodelling, have been proposed as new pharmacological therapeutic approaches. Potential upstream therapies for AF comprise a variety of agents such as angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB), statins, N-3 polyunsaturated fatty acids and steroids. On the basis of experimental data, clinical studies have provided information on the potential of upstream therapy for the prevention of AF across a broad spectrum of cardiovascular patient groups. In patients with heart failure or hypertension, data are sufficient to support the use of ACEI or ARB as treatment that may decrease the risk of AF beyond their other beneficial effects. Similarly, it is highly possible that the use of statin in patients with a recognized indication may be associated with a benefit against AF. However, in most clinical settings, the evidence appears to be insufficient to drive changes in therapy management per se, and large-scale, randomized controlled trials with adequately defined endpoints are still needed. The results from these trials may help to understand the complex mechanisms that lead to AF, and may clarify the benefit-to-risk ratio of these new therapeutic approaches.     

Taking the heart failure battle inside the cell: small molecule targeting of Gβγ subunits

Heart failure (HF) is devastating disease with poor prognosis. Elevated sympathetic nervous system activity and outflow, leading to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) signaling and responsiveness, are salient characteristic of HF progression. These pathologic effects on β-AR signaling and HF progression occur in part due to Gβγ-mediated signaling, including recruitment of receptor desensitizing kinases such as G-protein coupled receptor (GPCR) kinase 2 (GRK2) and phosphoinositide 3-kinase (PI3K), which subsequently phosphorylate agonistoccupied GPCRs. Additionally, chronic GPCR signaling signals chronically dissociated Gβγ subunits to interact with multiple effector molecules that activate various signaling cascades involved in HF pathophysiology. Importantly, targeting Gβγ signaling with large peptide inhibitors has proven a promising therapeutic paradigm in the treatment of HF. We recently described an approach to identify small molecule Gβγ inhibitors that selectively block particular Gβγ functions by specifically targeting a Gβγ protein-protein interaction "hot spot." Here we describe their effects on Gβγ downstream signaling pathways, including their role in HF pathophysiology. We suggest a promising therapeutic role for small molecule inhibition of pathologic Gβγ signaling in the treatment of HF. This article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure.”     

Caspase-dependent regulation of the ubiquitin–proteasome system through direct substrate targeting

Drosophila inhibitor of apoptosis (IAP) 1 (DIAP1) is an E3 ubiquitin ligase that regulates apoptosis in flies, in large part through direct inhibition and/or ubiquitinylation of caspases. IAP antagonists, such as Reaper, Hid, and Grim, are thought to induce cell death by displacing active caspases from baculovirus IAP repeat domains in DIAP1, but can themselves become targets of DIAP1-mediated ubiquitinylation. Herein, we demonstrate that Grim self-associates in cells and is ubiquitinylated by DIAP1 at Lys136 in an UbcD1-dependent manner, resulting in its rapid turnover. K48-linked ubiquitin chains are added almost exclusively to BIR2-bound Grim as a result of its structural proximity to DIAP1’s RING domain. However, active caspases can simultaneously cleave Grim at Asp132, removing the lysine necessary for ubiquitinylation as well as any existing ubiquitin conjugates. Cleavage therefore enhances the stability of Grim and initiates a feed-forward caspase amplification loop, resulting in greater cell death. In summary, Grim is a caspase substrate whose cleavage promotes apoptosis by limiting, in a target-specific fashion, its ubiquitinylation and turnover by the proteasome.Apoptosis, or programmed cell death, is broadly conserved throughout nature, from flies to humans (1). In most instances, the execution of apoptosis is carried out by cysteinyl aspartate-specific proteases (i.e., caspases) through proteolytic-based signal transduction pathways (2). Upstream initiator caspases, such as caspase-9 in humans and its paralogue Drosophila Nedd2-like caspase (DRONC) in flies, are first activated via their interactions with adapter proteins and in turn activate the downstream effector caspases, caspase-3 and Drosophila interleukin-1β–converting enzyme (DrICE), respectively (2, 3). Once activated, effector caspases are responsible for dismantling the cell through cleavage of literally hundreds of structural and regulatory proteins (4). Caspase cleavage can inactivate proteins or generate dominant-negative inhibitors, as in the case of gelsolin, RIP1, and eIF4E-BP1 (4). Moreover, caspase cleavage of numerous substrates, including IRF-3, ErbB2, cyclin E, claspin, SSRP1, and Twist, can enhance their turnover by the proteasome (5–10). Conversely, caspases can also constitutively activate proteins, particularly kinases such as PKC and Mst1 (11, 12), or change the function of a protein altogether, as seen in the conversion of antiapoptotic BCL-2 proteins into proapoptotic BAX-like proteins (13).Notably, even following the activation of caspases, inhibitor of apoptosis (IAP) proteins, such as X-linked IAP (XIAP) in mammals and DIAP1 in flies, can suppress apoptosis through inhibition of caspases (14–19). All IAPs contain baculovirus IAP repeat (BIR) domains and many possess RING and UBA domains, imparting them with E3 ubiquitin and NEDD8 ligase activity and the ability to bind polyubiquitin chains (20, 21). Thus, XIAP and DIAP1 directly bind and inhibit, ubiquitinylate, and/or neddylate initiator and effector caspases through distinct BIR domains (15, 16, 19, 22–24). In some circumstances, ubiquitinylation marks these enzymes for proteasomal degradation, whereas, in other cases, K63-based ubiquitinylation or neddylation fail to increase protein turnover but nevertheless inhibit protease activity through as yet ill-defined mechanisms (22, 25–27).Finally, a further level of regulation exists in the form of endogenous inhibitors of IAPs. These so-called “IAP antagonists” possess an IAP binding motif (IBM) through which they bind to IAPs and disrupt their interactions with caspases (28). Reaper, Hid, and Grim were the first IAP antagonists to be discovered in flies and were shown to regulate cell death during development, at least in part, by binding to DIAP1, displacing caspases, and inducing autoubiquitinylation and turnover of DIAP1 (18, 19, 29–35). In the present study, we have discovered that DIAP1, in conjunction with the E2 ubiquitin-conjugating enzyme UbcD1, polyubiquitinylates Grim through K48- but not K63-based linkages, resulting in increased Grim turnover. Grim self-associates in cells and binds to both the BIR1 and BIR2 domains in DIAP1, but only the BIR2-bound Grim is significantly ubiquitinylated by DIAP1 in a RING-dependent manner. More surprisingly, Grim is also cleaved by caspases at its C terminus, removing the only lysine residue present in this IAP antagonist. Following caspase cleavage, Grim still binds to DIAP1 but is no longer ubiquitinylated and therefore persists in cells, propagating the death signal through increased activation of caspases.