The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Improving performance of docking-based virtual screening by structural filtration

In the current study an innovative method of structural filtration of docked ligand poses is introduced and applied to improve the virtual screening results. The structural filter is defined by a protein-specific set of interactions that are a) structurally conserved in available structures of a particular protein with its bound ligands, and b) that can be viewed as playing the crucial role in protein-ligand binding. The concept was evaluated on a set of 10 diverse proteins, for which the corresponding structural filters were developed and applied to the results of virtual screening obtained with the Lead Finder software. The application of structural filtration resulted in a considerable improvement of the enrichment factor ranging from several folds to hundreds folds depending on the protein target. It appeared that the structural filtration had effectively repaired the deficiencies of the scoring functions that used to overestimate decoy binding, resulting into a considerably lower false positive rate. In addition, the structural filters were also effective in dealing with some deficiencies of the protein structure models that would lead to false negative predictions otherwise. The ability of structural filtration to recover relatively small but specifically bound molecules creates promises for the application of this technology in the fragment-based drug discovery.     

Identification of a novel proliferation-inducing determinant using lentiviral expression cloning

One of the major challenges in the post-genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation-modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation-inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP-expressing oncoretroviruses to target proliferation-competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd-uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today’s untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation-modulating genes is essential for developing new strategies for gene therapy and tissue engineering.     

The role of hydrophobic interactions on alcohol binding in the active center of penicillin acylases

Inhibition of penicillin acylases from Escherichia coli and Alcaligenes faecalis by aliphatic and aromatic alcohols was studied. It was shown that the inhibition of both enzymes has competitive nature and they bind the alcohols at the acyl group binding site of the enzyme active center. The free energy of alcohol sorption was shown to be linearly dependent on the hydrophobicity of the inhibitor with slopes of 1.6 and 1.7, demonstrating extremely effective hydrophobic interactions. To rationalize the observed distinctions in the inhibiting properties of aromatic and aliphatic alcohols beginning with butanol, it was suggested that the loss of entropy occurring on the interaction of the ligand with the tightly restricted hydrophobic pocket of the active center makes an essential contribution to the overall energetics of complex formation.     

啤酒酵母菌的细胞融合

啤酒多倍体酵母菌原生质体已成功地与单倍体原生质体进行融合。经细胞壁再生后,稳定的融合重组体被分离出来。这些融合体的基因分析表明,融合体中含有双亲的基因型。孢子形成良好,且每个子囊中含有四个孢子,每个孢子确实是二倍体。这样原生质体融合就提供了一个对啤酒酿造酵母进行遗传分析的方法。但是如果没有一个方便的杂交技术,这个方法将是很困难的。     

2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with K_i = 250 μM (95% CI: 106–405 μM) and dose-dependently increased the rate of the ATP hydrolysis with K_M = 112 μM (95% CI: 28–195 μM). The kinetic scheme consistent with this experimental data is proposed.     

TSAR,a new graph–theoretical approach to computational modeling of protein side‐chain flexibility: Modeling of ionization properties of proteins

A new graph–theoretical approach called thermodynamic sampling of amino acid residues (TSAR) has been elaborated to explicitly account for the protein side chain flexibility in modeling conformation‐dependent protein properties. In TSAR, a protein is viewed as a graph whose nodes correspond to structurally independent groups and whose edges connect the interacting groups. Each node has its set of states describing conformation and ionization of the group, and each edge is assigned an array of pairwise interaction potentials between the adjacent groups. By treating the obtained graph as a belief‐network—a well‐established mathematical abstraction—the partition function of each node is found. In the current work we used TSAR to calculate partition functions of the ionized forms of protein residues. A simplified version of a semi‐empirical molecular mechanical scoring function, borrowed from our Lead Finder docking software, was used for energy calculations. The accuracy of the resulting model was validated on a set of 486 experimentally determined pK_a values of protein residues. The average correlation coefficient (R) between calculated and experimental pK_a values was 0.80, ranging from 0.95 (for Tyr) to 0.61 (for Lys). It appeared that the hydrogen bond interactions and the exhaustiveness of side chain sampling made the most significant contribution to the accuracy of pK_a calculations. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.     

Sex differences in the rate of fatigue development and recovery

Background

Many musculoskeltal injuries in the workplace have been attributed to the repetitive loading of muscle and soft tissues. It is not disputed that muscular fatigue is a risk factor for musculoskeltal injury, however the disparity between gender with respect to muscular fatigability and rate of recovery is not well understood. Current health and safety guidelines do not account for sex differences in fatiguability and may be predisposing one gender to greater risk. The purpose of this study was to quantify the sex differences in fatigue development and recovery rate of lower and upper body musculature after repeated bouts of sustained isometric contractions.

Methods

Twenty-seven healthy males (n = 12) and females (n = 15) underwent bilateral localized fatigue of either the knee extensors (male: n = 8; female: n = 8), elbow flexors (male: n = 8; female: n = 10), or both muscle groups. The fatigue protocol consisted of ten 30-second sub-maximal isometric contractions. The changes in maximum voluntary contraction (MVC), electrically evoked twitches, and motor unit activation (MUA) were assessed along with the ability to control the sustained contractions (SLP) during the fatigue protocol using a mixed four-factor repeated measures ANOVA (gender × side × muscle × time) design with significance set at p < 0.05.

Results

There was a significant loss of MVC, MUA, and evoked twitch amplitude from pre- to post-fatigue in both the arms and legs. Males had greater relative loss of isometric force, a higher rate of fatigue development, and were less capable of maintaining the fatiguing contractions in the legs when compared to the females.

Conclusion

The nature of the induced fatigue was a combination of central and peripheral fatigue that did not fully recover over a 45-minute period. The results appear to reflect sex differences that are peripheral, and partially support the muscle mass hypothesis for explaining differences in muscular fatigue.

     

Is low dose inhaled corticosteroid therapy as effective for inflammation and remodeling in asthma? A randomized,parallel group study

Background

While most of the clinical benefits of inhaled corticosteroid (ICS) therapy may occur at low doses, results of dose-ranging studies are inconsistent. Although symptom/lung function response to low and high dose ICS medication is comparable, it is uncertain whether low dose ICSs are as effective as high dose in the treatment of inflammation and remodeling.

Methods

22 mild or moderate asthmatic adult subjects (corticosteroid free for > 2 months) participated in a randomized, parallel group study to compare effects of fluticasone propionate (FP) 200 mcg/day and 1000 mcg/day. Alveolar macrophage (AM)-derived cytokines and basement membrane thickness (BMT) were measured at baseline and after 7 weeks treatment while symptoms, spirometry, exhaled nitric oxide (eNO) and airway hyperresponsiveness (AHR) to mannitol at baseline and 6 weeks.

Results

FP improved spirometry, eNO, symptoms and AHR with no difference between low and high dose FP. Both high and low dose FP reduced GM-CSF, TNF-alpha and IL-1ra, with no change in BMT and with no differences between low and high dose FP.

Conclusions

200 μg/day of FP was as effective as 1000 μg/day in improving asthma control, airway inflammation, lung function and AHR in adults in the short term. Future studies should examine potential differential effects between low and high dose combination therapy (ICS/long acting beta agonist) on inflammation and airway remodeling over longer treatment periods.