Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.Subject terms: Virology, Molecular evolution     

Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase

A highly thermostable xylanase (Xyl I) produced by Thermomonospora sp. was purified to homogeneity and was classified as a family 10 xylanase based on its molecular weight (38,000 Da) and isoelectric point (4.1). K2d analysis showed that the secondary structure of Xyl I was made up of 38% alpha-helix and 10% beta-sheet. The optimal temperature for the activity of Xyl I was 80 degrees C. Xyl I was highly thermostable with half-lives of 86, 30, and 15 min at 80, 90, and 100 degrees C respectively. Xyl I was stable in an expansive pH range of 5 to 10 with more than 75% residual activity. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent chemoaffinity labeling and trinitrobenzenesulphonic acid (TNBS) as chemical modifier have revealed the presence of a single lysine residue in the active site of Xyl I. The high pK value for the basic limb of the pH profile reflects the ionization of a lysine residue. The higher K(m) values and similar k(cat) values of the TNBS modified enzyme in comparison to native enzyme and the substrate protection against OPTA and TNBS, suggested the presence of the lysine residue in the substrate-binding site.     

Calling behavior and directional hybridization between two toads (Bufo microscaphus x B. woodhousii) in Arizona

Male calling effort and mitochondrial DNA (mtDNA) variation were examined in a breeding chorus of toads from a hybrid zone between Bufo microscaphus and B. woodhousii in central Arizona. The chorus comprised 50 B. microscaphus and 17 hybrids, identified on the basis of morphology and advertisement calls; no pure B. woodhousii were observed. Males produced advertisement calls throughout the early evening, even when relatively large numbers of males (>50) were present at the chorus; active searching and satellite tactics were not observed. Calling efforts (call duration x call rate) of hybrids (23.9%, n = 8) and B. microscaphus (24.9%, n = 19) were similar and comparable to call efforts of B. woodhousii (21.9%, n = 10) from a different site. Moreover, repeatabilities of calling effort were significant (r = 0.45) for hybrid males, but not for B. microscaphus and B. woodhousii. Thus, calling behavior of hybrid males was neither significantly reduced nor more variable than that of their parental species. The distribution of mtDNA haplotypes revealed directional introgression is occurring between male B. microscaphus and female B. woodhousii: All 17 hybrids possessed B. woodhousii mtDNA. The proximate mechanism driving hybridization appears to involve common male (B. microscaphus) and rare female (B. woodhousii) matings as B. woodhousii expands its range.     

Deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement

The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail- or FDelta19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail- mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail- mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail- is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.     

15-Lipoxygenase-1 has anti-tumorigenic effects in colorectal cancer

The localization of 15-lipoxygenase-1 (15-LO-1) in human colorectal carcinoma and normal adjacent tissue was examined using immunohistochemistry. In normal tissues, 15-LO-1 was strongly localized in the mucosal epithelium. Conversely, in tumor tissues, staining for 15-LO-1 was dispersed throughout the tissue, weak in neoplastic epithelium, and strong in stromal inflammatory cells. The addition of 50 microM 13(S)-hydroxyeicosatetraenoic acid (HODE), resulted in decreased cell proliferation after 72 h, but lower concentrations (5 or 10 microM) had no effect compared to vehicle treated Caco-2 cells. In addition, 13(S)-HODE had no effect on apoptosis or differentiation of the Caco-2 cells. Microarray analyses of RNA from Caco-2 cells treated with 5 microM 13(S)-HODE revealed changes in 17 genes. HCT-116 colorectal cells were stably transfected with 15-LO-1. In athymic nude mice, transplantable tumors derived from 15-LO-1 HCT-116 cells were smaller than tumors derived from vector HCT-116 cells. These data demonstrate that 13(S)-HODE induces changes in gene expression and has anti-tumorigenic effects.     

Effect of creatine on contractile force and sensitivity in mechanically skinned single fibers from rat skeletal muscle

Increasing the intramuscular stores of total creatine [TCr = creatine (Cr) + creatine phosphate (CrP)] can result in improved muscle performance during certain types of exercise in humans. Initial uptake of Cr is accompanied by an increase in cellular water to maintain osmotic balance, resulting in a decrease in myoplasmic ionic strength. Mechanically skinned single fibers from rat soleus (SOL) and extensor digitorum longus (EDL) muscles were used to examine the direct effects on the contractile apparatus of increasing [Cr], increasing [Cr] plus decreasing ionic strength, and increasing [Cr] and [CrP] with no change in ionic strength. Increasing [Cr] from 19 to 32 mM, accompanied by appropriate increases in water to maintain osmolality, had appreciable beneficial effects on contractile apparatus performance. Compared with control conditions, both SOL and EDL fibers showed increases in Ca²⁺ sensitivity (+0.061 ± 0.004 and +0.049 ± 0.009 pCa units, respectively) and maximum Ca²⁺-activated force (to 104 ± 1 and 105 ± 1%, respectively). In contrast, increasing [Cr] alone had a small inhibitory effect. When both [Cr] and [CrP] were increased, there was virtually no change in Ca²⁺ sensitivity of the contractile apparatus, and maximum Ca²⁺-activated force was 106 ± 1% compared with control conditions in both SOL and EDL fibers. These results suggest that the initial improvement in performance observed with Cr supplementation is likely due in large part to direct effects of the accompanying decrease in myoplasmic ionic strength on the properties of the contractile apparatus. ergogenic aid; muscle contraction; fatigue     

The activities of cyclin D1 that drive tumorigenesis

The proto-oncogene cyclin D1 has been implicated in the genesis of a large proportion of human tumors from diverse histological origins. It has long been assumed that the action of cyclin D1, as an activator of cdk4 and cdk6 and leading to progression through the G1 phase of the cell cycle, underlies its pathological activity. But, more recently, analyses of the patterns of gene expression in human cancer have revealed a previously unappreciated mechanism of action for cyclin D1, suggesting that both cdk-dependent and cdk-independent activities might contribute to tumorigenesis. The development of therapeutics designed to target the aberrant activity of cyclin D1 in human cancers will rely upon an intimate molecular understanding of these distinct mechanisms of actions and their relative importance. Here, we describe the known functions of the cyclin D1 oncogene and delineate the evidence that cdk-independent actions are important for cyclin D1-mediated oncogenesis.