Cytochemical and electron microscopic demonstration of organelle translocation and the inhibition of such translocation by colchicine during the mitosis of rat hepatocytes

Hepatocyte lysosomes, mitochondria, and peroxisomes show a dramatic translocation during mitosis induced by partial hepatectomy. During prophase, all three organelles move to the perinuclear cytoplasm. In metaphase, they become concentrated in the polar regions. During telophase, these organelles form clusters in the juxtanuclear regions. This organelle translocation is inhibited by the administration of a low concentration of colchicine, suggesting an involvement of microtubules in their movement.     

Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins

Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.     

Quinine and quinidine production by cinchona leaf,root and unorganized cultures

Serially propagated Cinchona ledgeriana and C. succirubra (Rubiaceae) leaf, root and unorganized suspension cultures established from germinated seeds were studied for quinine and quinidine production. Leaf organ cultures were grown and subcultured in Murashige and Skoog's Revised Tobacco Medium supplemented with benzyladenine; root organ cultures were grown on the same medium supplemented with indolebutyric acid; and unorganized suspension cultures were grown on the same medium supplemented with 2,4-dichlorophenoxyacetic acid and benzyladenine. On a dry weight basis, leaf organ cultures of C. ledgeriana contained 0.06 % quinine and 0.05 % quinidine and of C. succirubra contained 0.04 % quinine and 0.04 % quinidine. No quinine and quinidine were detected in either root organ or unorganized suspension cultures.     

Acetyl groups in cell-wall preparations from higher plants

O-Acetyl groups were detected by i.r. spectroscopy in cell-wall preparations from grasses and other higher plants and their presence was confirmed chemically. The amounts present are likely to influence both the physical state of the cell-wall polysaccharides and also their digestion by enzymes.     

CellDynaMo–stochastic reaction-diffusion-dynamics model: Application to search-and-capture process of mitotic spindle assembly

We introduce a Stochastic Reaction-Diffusion-Dynamics Model (SRDDM) for simulations of cellular mechanochemical processes with high spatial and temporal resolution. The SRDDM is mapped into the CellDynaMo package, which couples the spatially inhomogeneous reaction-diffusion master equation to account for biochemical reactions and molecular transport within the Langevin Dynamics (LD) framework to describe dynamic mechanical processes. This computational infrastructure allows the simulation of hours of molecular machine dynamics in reasonable wall-clock time. We apply SRDDM to test performance of the Search-and-Capture of mitotic spindle assembly by simulating, in three spatial dimensions, dynamic instability of elastic microtubules anchored in two centrosomes, movement and deformations of geometrically realistic centromeres with flexible kinetochores and chromosome arms. Furthermore, the SRDDM describes the mechanics and kinetics of Ndc80 linkers mediating transient attachments of microtubules to the chromosomal kinetochores. The rates of these attachments and detachments depend upon phosphorylation states of the Ndc80 linkers, which are regulated in the model by explicitly accounting for the reactions of Aurora A and B kinase enzymes undergoing restricted diffusion. We find that there is an optimal rate of microtubule-kinetochore detachments which maximizes the accuracy of the chromosome connections, that adding chromosome arms to kinetochores improve the accuracy by slowing down chromosome movements, that Aurora A and kinetochore deformations have a small positive effect on the attachment accuracy, and that thermal fluctuations of the microtubules increase the rates of kinetochore capture and also improve the accuracy of spindle assembly.     

Genetic and environmental influences on migraine: a twin study across six countries.

Migraine is a common neurovascular brain disorder that is manifested in recurrent episodes of disabling headache. The aim of the present study was to compare the prevalence and heritability of migraine across six of the countries that participate in GenomEUtwin project including a total number of 29,717 twin pairs. Migraine was assessed by questionnaires that differed between most countries. It was most prevalent in Danish and Dutch females (32% and 34%, respectively), whereas the lowest prevalence was found in the younger and older Finnish cohorts (13% and 10%, respectively). The estimated genetic variance (heritability) was significant and the same between sexes in all countries. Heritability ranged from 34% to 57%, with lowest estimates in Australia, and highest estimates in the older cohort of Finland, the Netherlands, and Denmark. There was some indication that part of the genetic variance was non-additive, but this was significant in Sweden only. In addition to genetic factors, environmental effects that are non-shared between members of a twin pair contributed to the liability of migraine. After migraine definitions are homogenized among the participating countries, the GenomEUtwin project will provide a powerful resource to identify the genes involved in migraine.     

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the α5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.     

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae – Identification of LYS228

Metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of β-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine β-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of β-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).