An essential cell cycle-regulated nucleolar protein relocates to the mitotic spindle where it is involved in mitotic progression in Trypanosoma brucei

TbNOP86 and TbNOP66 are two novel nucleolar proteins isolated in Trypanosoma brucei. They share 92.6% identity, except for an additional C-terminal domain of TbNOP86 of 182 amino acids in length. Both proteins are found in Trypanosomatidae, but similarity to other eukaryotic proteins could not be found. TbNOP86 and TbNOP66 are expressed at similar level in procyclic and bloodstream forms, although the relative level of expression of TbNOP66 is 11 times lower. TbNOP86 undergoes post-translational modifications, as it is found predominantly at 110 kDa compared with the predicted 86 kDa. Immunofluorescence of overexpressed ty-tagged TbNOP86 and TbNOP66 showed that both proteins accumulated in the nucleolus of G(1) cells. This was confirmed by the co-localization of an endogenous TbNOP86-myc with the nucleolar protein Nopp140. TbNOP86-ty localization is cell cycle-regulated, because it colocalizes with the mitotic spindle in mitotic cells. TbNOP86 is required for mitotic progression in both life stages as depleted cells are enriched in the G(2)/M phase. In procyclic cells, a reduced growth rate is accompanied by an accumulation of zoids (0N1K), 2N1K, and multinucleated cells (xNyK). The 2N1K cells are blocked in late mitosis as nucleolar segregation is completed. TbNOP86 depletion in bloodstream form caused a drastic growth inhibition producing cells bearing two kinetoplasts and an enlarged nucleus (1N(*)2K), followed by an accumulation of 2N2K cells with connected nuclei and xNyK cells. These studies of TbNOP86 provide a more comprehensive account of proteins involved in mitotic events in trypanosomes and should lead to the identification of partners with similar function.     

Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart

Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 mug/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 mug/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 +/- 3.5% (vehicle) to 11.0 +/- 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 +/- 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.     

Discovery of novel 2,3-diarylfuro[2,3-b]pyridin-4-amines as potent and selective inhibitors of Lck: synthesis, SAR, and pharmacokinetic properties

2,3-Diarylfuro[2,3-b]pyridine-4-amines are a novel class of potent and selective inhibitors of Lck. The discovery, synthesis, and structure activity relationships of this series of inhibitors are reported. The most promising compounds were also profiled to deduce their pharmacokinetic properties.     

Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces

AIM: To evaluate US Centers for Disease Control and Prevention recommended swab surface sample collection method for recovery efficiency and limit of detection for powdered Bacillus spores from nonporous surfaces. METHODS AND RESULTS: Stainless steel and painted wallboard surface coupons were seeded with dry aerosolized Bacillus atrophaeus spores and surface concentrations determined. The observed mean rayon swab recovery efficiency from stainless steel was 0.41 with a standard deviation (SD) of +/-0.17 and for painted wallboard was 0.41 with an SD of +/-0.23. Evaluation of a sonication extraction method for the rayon swabs produced a mean extraction efficiency of 0.76 with an SD of +/-0.12. Swab recovery quantitative limits of detection were estimated at 25 colony forming units (CFU) per sample area for both stainless steel and painted wallboard. CONCLUSIONS: The swab sample collection method may be appropriate for small area sampling (10 -25 cm2) with a high agent concentration, but has limited value for large surface areas with a low agent concentration. The results of this study provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequence of an inappropriate public health response founded on information gathered using an ineffective or unreliable sample collection method has the potential for undesired social and economic impact.     

Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.     

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a β(-) emitter such as (90)Y with an optical imager.     

Effects of the new arginase inhibitor N(omega)-hydroxy-nor-L-arginine on NO synthase activity in murine macrophages.

In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, l-arginine. The objectives of this study were (i) to test the new alpha-amino acid N(omega)-hydroxy-nor-l-arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on l-arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N(omega)-hydroxy-l-arginine (NOHA), an intermediate in the l-arginine/NO pathway, to inhibit the hydrolysis of l-arginine to l-ornithine catalyzed by unstimulated murine macrophages (IC(50) values 12 +/- 5 and 400 +/- 50 microM, respectively). Stimulation of murine macrophages with interferon-gamma and lipopolysaccharide (IFN-gamma + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-gamma + LPS-stimulated macrophage (IC(50) value 10 +/- 3 microM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and l-citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on l-arginine availability.     

Nutritional parameters modify muricidal behavior of male Wistar rats: preventive effects of amino acids and 4' Cl diazepam.

In male Wistar rats fed a diet enriched in polyunsaturated fatty acids and starch (PUFA+S), the percentage of muricidal (Mu) rats increased to 82% within 60 days. Mu rats had higher serum triglyceride levels and lower cholesterol levels than non-Mu rats. Water intake decreased in all rats on the PUFA+S diet concurrently with the increase in the proportion of Mu rats; protracted water restriction in rats fed standard diet also increased the percentage of Mu rats. In the offspring of two Wistar females fed the PUFA+S diet, the proportion of young Mu rats was 67%. When the PUFA+S diet was replaced with standard diet, the induced Mu behavior was not reversed. PK11195 (6 mg/kg i.p.), clonazepam (0.2 mg/kg i.p.), and flumazenil (15 mg/kg i.p.) were ineffective in reversing the induced Mu behavior, whereas 4'-chlorodiazepam (5 mg/kg i.p.) or muscimol (0.5 mg/kg i.p.) caused reversals of 63% or 50%, respectively. A 5-hydroxytryptophan overload (60 mg/kg i.p.) also reversed Mu behavior by 71%. All reversal effects were temporary. Pretreatment with yeast for 7 days before the PUFA+S diet was given prevented induction for more than 90 days on the PUFA+S diet, while similar pretreatment 4'Cl-diazepam resulted in 71% prevention of induction. The results are analyzed in terms of the involvement of endozepin, vasopressin, and serotonin receptors, and of possible genetic parameters.