Identification of long-lived proteins in the mitochondria reveals increased stability of the electron transport chain

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Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.     

Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

The impact of tick parasites on the behaviour of the lizard Tiliqua rugosa

Populations of the Australian sleepy lizard, Tiliqua rugosa, near Mt. Mary, South Australia carry natural infestations of two tick species Aponomma hydrosauri and Amblyomma limbatum. In field experiments at two sites, 18 km apart, lizards with experimentally increased tick loads had smaller home ranges, moved shorter distances in a day, and were found basking more but moving less often than lizards from which ticks were experimentally removed. The results were consistent for adult lizards in two years, and for sub-adults in a third year. Laboratory trials showed that juvenile lizards that had tick infestations had lower sprint speeds than uninfested siblings, and that adults with tick infestations had less endurance than those that were uninfested. The results contrast with those of a previous survey that showed that lizards with high tick loads had greater body size and remained longer at a site, but indicate that there may be a balance, for lizards, between the fitness advantages in occupying habitats with high-quality resources, and the costs from parasites that also prefer those habitats. Received: 02 March 1999 / Accepted: 07 October 1999     

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses.