Large-scale purification of recombinant human angiostatin

A process for the purification of recombinant human angiostatin (rhAngiostatin), produced by Pichia pastoris fermentation operated at the 2000-L scale, is reported. rhAngiostatin was recovered and purified directly from crude fermentation broth by cation exchange expanded bed adsorption chromatography. Anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography were used for further purification. Full-length rhAngiostatin was separated from rhAngiostatin molecules fragmented by endoproteolysis. On average, 140 g of rhAngiostatin was produced per batch, with an overall yield of 59% (n = 9). The purification process was completed in approximately 48 h and used only inexpensive and nontoxic raw materials. Methods development, process synthesis, and process scale-up data are presented and discussed.     

Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection

Recent thymic emigrants can be identified by T cell receptor excision circles (TRECs) formed during T-cell receptor rearrangement. Decreasing numbers of TRECs have been observed with aging and in human immunodeficiency virus (HIV)-1 infected individuals, suggesting thymic impairment. Here, we show that in healthy individuals, declining thymic output will affect the TREC content only when accompanied by naive T-cell division. The rapid decline in TRECs observed during HIV-1 infection and the increase following HAART are better explained not by thymic impairment, but by changes in peripheral T-cell division rates. Our data indicate that TREC content in healthy individuals is only indirectly related to thymic output, and in HIV-1 infection is mainly affected by immune activation.     

Loss of the precise control of photosynthesis and increased yield of non‐radiative dissipation of excitation energy after mild heat treatment of barley leaves

The after effects of a short exposure of intact barley leaves to moderately elevated temperature (40°C, 5 min) on the induction transients and the irradiance dependencies of photosynthesis and chlorophyll fluorescence are presented. This mild heat treatment strongly reduced the oscillations in the rate of photosynthesis and in the yield of chlorophyll fluorescence. However, only a 25% irreversible inhibition of maximum photosynthetic capacity of photosystem II (PSII) measured by oxygen evolution was produced and the intrinsic quantum yield of PSII measured by the chlorophyll fluorescence ratio (F_m‐ F_o)/F_m decreased by only 15%. In contrast, the above treatment increased radiationless dissipation processes in PSII by a factor of two. In heat‐treated leaves, photosynthesis was not saturated even by strong light. Both ΔpH‐dependent quenching of excitons in PSII (including formation of zeaxanthin) and state 1/state 2 transition were found to be stimulated. Heat exposure enhanced the control of PSII activity by PSI, as evidenced by a significant increase in the quenching effect of far‐red light on the maximum yield of chlorophyll fluorescence. It was deduced that after mild heat treatment, the photosynthetic apparatus in leaves lacks the precise coordinating control of electron transport and carbon metabolism owing to the inability of PSII to support electron transport at a level adequate for carbon metabolism. This effect was not related to the small irreversible thermal damage to PSII, but was rather due to a significant increase in non‐photochemical quenching of excitation energy.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Bacterial community changes during bioremediation of aliphatic hydrocarbon-contaminated soil

The microbial community response during the oxygen biostimulation process of aged oil-polluted soils is poorly documented and there is no reference for the long-term monitoring of the unsaturated zone. To assess the potential effect of air supply on hydrocarbon fate and microbial community structure, two treatments (0 and 0.056 mol h?1 molar flow rate of oxygen) were performed in fixed bed reactors containing oil-polluted soil. Microbial activity was monitored continuously over 2 years throughout the oxygen biostimulation process. Microbial community structure before and after treatment for 12 and 24 months was determined using a dual rRNA/rRNA gene approach, allowing us to characterize bacteria that were presumably metabolically active and therefore responsible for the functionality of the community in this polluted soil. Clone library analysis revealed that the microbial community contained many rare phylotypes. These have never been observed in other studied ecosystems. The bacterial community shifted from Gammaproteobacteria to Actinobacteria during the treatment. Without aeration, the samples were dominated by a phylotype linked to the Streptomyces. Members belonging to eight dominant phylotypes were well adapted to the aeration process. Aeration stimulated an Actinobacteria phylotype that might be involved in restoring the ecosystem studied. Phylogenetic analyses suggested that this phylotype is a novel, deep-branching member of the Actinobacteria related to the well-studied genus Acidimicrobium.     

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.     

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.