Actin acetylation in Drosophila tissue culture cells

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.This work was supported by a grant from the NIH (GM 22866).     

Five-year perspective of the large-scale growth of mammalian cells in suspension culture

The Cell Culture Center at the University of Alabama in Birmingham was set up to produce large quantities of cells and their products from suspension cultured cell lines. This system has now been in operation for over five years and has been effective in producing large quantities of mammalian cells of murine and human origin. This article describes the system and some growth parameters which have been of importance for large-scale mammalian cell growth.     

Iron uptake with ferripyochelin and ferric citrate by Pseudomonas aeruginosa.

Pyochelin is an iron-binding compound produced by Pseudomonas aeruginosa and demonstrates siderophore activity by its involvement in iron transport. During the transport process, an energy-independent association of [55Fe]ferripyochelin with bacteria occurred within the initial 30 s of reaction, followed by an energy-dependent accumulation of iron. The energy-independent association with iron appeared to be at the surface of the bacteria because the iron could be washed from the cells with thioglycolate, whereas accumulated iron was not washed from the bacteria. Energy-independent association of iron with bacteria and energy-dependent accumulation of iron in the presence of ferripyochelin varied concomitantly in cells grown under various conditions, but pyochelin synthesis appeared to be controlled separately. 55Fe complexed with citrate was also taken up by P. aeruginosa with a lower level of initial cell association. Bacterial mechanisms for iron uptake from ferric citrate were present in cells grown in a variety of media and were in lowest levels in cells grown in citrate. The synthesis of bacterial components for iron uptake from ferric citrate and from ferripyochelin was inhibited by high concentrations of iron supplied in growth media.     

Sexually dimorphic expression of secreted frizzled-related (SFRP) genes in the developing mouse Müllerian duct

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.     

Lipase-mediated purification of methyl nonactate, an important natural product building block for diversity-oriented synthesis

Methyl nonactate is a valuable starting material for the production of natural product-like combinatorial libraries and ketide amino acids, a series of novel conformationally constrained amino acid analogues. Fermentation of Streptomyces griseus generates high titers of macrotetrolide antibiotics from which methyl nonactate can be generated by methanolysis. Unfortunately, this approach generates mixtures of homologues that are not economically separable. We report a specific lipase-mediated hydrolysis of nonactate derivatives that discriminates between homologues and is the foundation of an economically tractable preparation of methyl nonactate in scale.     

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Porcine respiratory coronavirus (PRCV) potentiates respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV (n = 34) or saline (n = 5) and euthanized 1-15days post inoculation (DPI). Virus was detected in the lungs from 1 to 9DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14+ monocyte-macrophages from 1 to 12DPI and CD14+ type 2 pneumocytes from 7 to 9DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1-12DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (x4.5) at 7DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.     

Will my protein crystallize? A sequence‐based predictor

We propose a machine-learning approach to sequence-based prediction of protein crystallizability in which we exploit subtle differences between proteins whose structures were solved by X-ray analysis [or by both X-ray and nuclear magnetic resonance (NMR) spectroscopy] and those proteins whose structures were solved by NMR spectroscopy alone. Because the NMR technique is usually applied on relatively small proteins, sequence length distributions of the X-ray and NMR datasets were adjusted to avoid predictions biased by protein size. As feature space for classification, we used frequencies of mono-, di-, and tripeptides represented by the original 20-letter amino acid alphabet as well as by several reduced alphabets in which amino acids were grouped by their physicochemical and structural properties. The classification algorithm was constructed as a two-layered structure in which the output of primary support vector machine classifiers operating on peptide frequencies was combined by a second-level Naive Bayes classifier. Due to the application of metamethods for cost sensitivity, our method is able to handle real datasets with unbalanced class representation. An overall prediction accuracy of 67% [65% on the positive (crystallizable) and 69% on the negative (noncrystallizable) class] was achieved in a 10-fold cross-validation experiment, indicating that the proposed algorithm may be a valuable tool for more efficient target selection in structural genomics. A Web server for protein crystallizability prediction called SECRET is available at http://webclu.bio.wzw.tum.de:8080/secret.     

Phylogenetic inferences in the dung-moss family Splachnaceae from analyses of cpDNA sequence data and implications for the evolution of entomophily

The moss family Splachnaceae is characterized by half of its members relying on insects for spore dispersal. These species grow on dung or other animal substrates. They produce small and aggregated spores, and their capsule is modified to attract coprophilous insects or carrion flies using olfactory and visual cues. Systematic concepts and implicit evolutionary inferences have relied much on variation in characters associated with the spore dispersal syndrome. Phylogenetic reconstructions based on sequence variation of two chloroplast loci (trnL-trnF region and the rps4 gene) suggest that most supraspecific taxa are poly- or paraphyletic. Transformations in morphological characters associated to the syndrome thus offer little if any phylogenetically informative signal. Brachymitrion is resolved in a nested position within Tayloria. A new combination, Tayloria immersa (Goffinet) Goffinet, Shaw & Cox is proposed for B. immersum. Only one of the five subgenera of Tayloria (subg. Orthodon) is potentially monophyletic. Voitia shares a common ancestor with Tetraplodon and is thus nested within the Splachnoideae. The affinities of Aplodon remain ambiguous. Reconstruction of shifts between wind and insect spore dispersal syndromes suggests that entomophily arose more than once and may have been followed by a reversal to the generalist strategy in two lineages.