CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress.

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Immediate early responses of avian tracheal epithelial cells to infection with highly pathogenic avian influenza virus.

Highly pathogenic (HP) avian influenza viruses (AIV) present an ongoing threat to the world poultry industry. In order to develop new AIV control strategies it is necessary to understand the underlying mechanism of viral infection at mucosal respiratory sites. Chicken and duck tracheal epithelial cells systems (TEC) were developed to study early host responses to AIV infection on TEC. Infection of 2 week-old chickens and ducks with the highly pathogenic AIV H5N1 Ck/Hong Kong/220/97 and Egret/Hong Kong/757.2/02 viruses together with TEC early responses to infection suggest the induction of differential innate immune responses. Growth curves indicated that although chicken and ducks TEC supported viral replication and re-infection, the capacity of the two viruses to replicate was not equal. A 42K probes chicken microarray system used to characterize differences in gene expression between chicken tracheal epithelial cells infected with these two highly pathogenic AIV identified expression of virus-specific molecular markers. The existence of dissimilar patterns of host gene expression as early as six hours post infection suggests that the differential growth characteristics of the two highly pathogenic AIV in tracheal epithelial cells is preceded by distinct host responses.

Telemetric implantable pressure sensor for short- and long-term monitoring of intracranial pressure.

Patients with hydrocephalus, idiopathic intracranial hypertension and head injury frequently require monitoring of intracranial pressure (ICP) and may need repeated episodes of monitoring months or years apart. The gold standard for measurement of ICP remains the external ventricular catheter. This is a fluid-filled catheter transducer system that allows regular recalibration and correction of zero drift by its position relative to a fixed anatomical reference. It also allows drainage of cerebrospinal fluid (CSF), providing a means of lowering the ICP. Several catheter tip transducer systems are currently in clinical use, including using strain gauges or fiber-optical pressure sensing techniques. In these devices, zero drift and calibration cannot be checked in vivo. All the ICP monitoring devices in current clinical use require a physical connection between the brain and the external environment. This is a source of infection and limits the duration of monitoring. A number of telemetric monitoring devices, in which data is in some way transmitted transcutaneously, have been developed over the last twenty years, but significant technical problems have precluded their use in routine clinical practice. All current ICP monitors are temporary percutaneous implanted devices. Placement of these devices carries significant morbidity, particularly infection. Patients undergoing repeated monitoring require multiple surgical procedures. Apart from decreasing the risk of infection in patients with severe head injury, the clinical value of an accurate telemetric ICP monitoring system which maintains its reliability over a long period of implantation is high.

Antimicrobial activity of some Hypericum species.

The crude methanolic extracts of six species of Hypericum [H. caprifoliatum Cham. & Schlecht., H. carinatum Griseb., H. connatum Lam., H. ternum A. St. Hil., H. myrianthum Cham. & Schlecht. and H. polyanthemum Klotzsch ex Reichardt] growing in southern Brazil were analyzed for antimicrobial activity against several microorganisms (bacteria and fungi). The most active plant was H. caprifoliatum, which showed activity against Staphylococcus aureus. Only H. polyanthemum and H. ternum extracts were active against Bacillus subtilis. None of the crude methanolic extracts showed activity against S. epidermidis, Escherichia coli or Saccharomyces cerevisiae. Extracts from these species were evaluated chemically and tannin, flavonoid and phenolic acids were the prominent compounds. The plants contained quercitrin, hyperoside (except H. connatum) and, less frequently, isoquercitrin and chlorogenic acid. In contrast to H. perforatum, which has high concentrations of rutin, these species do not produce this flavonoid or it appears as traces. The tannin concentration varied between 5.1 and 16.7% in H. myrianthum and H. ternum, respectively.

Control of the arabinose regulon in Bacillus subtilis by AraR in vivo: crucial roles of operators, cooperativity, and DNA looping.

The proteins involved in the utilization of L-arabinose by Bacillus subtilis are encoded by the araABDLMNPQ-abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene, and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within the araABDLMNPQ-abfA (OR(A1) and OR(A2)) and araE (OR(E1) and OR(E2)) promoters and noncooperatively to a single operator in the araR (OR(R3)) promoter region. Here, we have investigated how AraR controls transcription from the ara regulon in vivo. A deletion analysis of the ara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes. Furthermore, OR(E1)-OR(E2) and OR(R3) are auxiliary operators for the autoregulation of araR and the repression of araE, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from the araABDLMNPQ-abfA operon and araE promoters, strongly suggesting that it is the major protein involved in the repression mechanism of L-arabinose-inducible expression in vivo. The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.