The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor.

The leukemogenic membrane glycoprotein of Friend spleen focus-forming virus (SFFV) has an apparent Mr of 55,000 (gp55), is encoded by a recombinant env gene, and occurs on cell surfaces and in intracellular organelles. There is evidence that the amino-terminal region of gp55 forms a dualtropic-specific domain that is connected to the remainder of the glycoprotein by a proline-rich linker (C. Machida, R. Bestwick, B. Boswell, and D. Kabat, Virology 144:158-172, 1985). Using the colinear form of a cloned polycythemic strain of SFFV proviral DNA, we constructed seven in-phase env mutants by insertion of linkers and by a deletion. The mutagenized SFFVs were transfected into fibroblasts and were rescued by superinfection with a helper murine leukemia virus. Four of the mutants cause erythroblastosis. These include one with a 6-base-pair (bp) insert in the ecotropic-related sequence near the 3' end of the gene, two with a 12- or 18-bp insert in the region that encodes the proline-rich linker, and one with a 6-bp insert in the dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific sequences that are highly conserved among strains of SFFV. A pathogenic revertant (RI-rev) was isolated from one mouse that developed erythroblastosis 3 weeks after infection with RI. RI-rev contains a second-site env mutation that affects the same domain as the primary mutation does and that increases the size of the encoded glycoprotein. All pathogenic SFFVs encode glycoproteins that are expressed on cell surfaces, whereas the nonpathogenic glycoproteins are exclusively intracellular. The pathogenic SFFVs also specifically cause a weak interference to superinfection by dualtropic MuLVs. These results are compatible with the multidomain model for the structure of gp55 and suggest that processing of gp55 to plasma membranes is required for pathogenesis. The amino-terminal region of gp55 binds to dualtropic murine leukemia virus receptors, and this interaction is preserved in the SFFV mutants that cause erythroblastosis.     

Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to an hrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s. pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn²⁺/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H₂O₂ in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.     

Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, <Emphasis Type="Italic">Butyrivibrio fibrisolvens</Emphasis>

Background  

Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA.     

Antenatal Screening for Down Syndrome Using Serum Placental Growth Factor with the Combined,Quadruple, Serum Integrated and Integrated Tests

Objective

To estimate the value of first or second trimester placental growth factor (PlGF) as an additional antenatal screening marker for Down syndrome.

Design

Nested case-control study.

Setting

Antenatal screening service.

Population or Sample

532 Down syndrome pregnancies and 1,155 matched unaffected pregnancies.

Methods

Stored maternal serum samples (−40°C) were assayed for PlGF. Monte Carlo simulation was used to estimate the screening performance of PlGF with the Combined, Quadruple, serum Integrated and Integrated tests.

Main Outcome Measures

Median PlGF levels in affected and unaffected pregnancies and screening performance (detection rates [DR] for specified false-positive rates [FPR] and vice versa).

Results

First trimester median PlGF was 15%, 28% and 39% lower in Down syndrome than unaffected pregnancies at 11, 12 and 13 completed weeks’ gestation respectively (all p<0.001). Second trimester median PlGF was 31% lower at 14 weeks (p<0.001), and the difference decreased (6% lower at 17 weeks). At a 90% DR with first trimester markers measured at 13 weeks, adding PlGF decreased the FPR from 11.1 to 5.1% using the Combined test, 9.3% to 4.5% using the serum Integrated test, and 3.4% to 1.5% using the Integrated test (or 1.5 to 1.4% with first trimester markers measured at 11 weeks). Adding PlGF to the Quadruple test (measured at 15 weeks) decreased the FPR from 10.0% to 9.6% at a 90% DR.

Conclusions

First trimester PlGF measurements improve the performance of antenatal screening for Down syndrome using the Combined, serum Integrated and Integrated tests. Second trimester PlGF measurements are of limited value.     

Pterosaur dietary hypotheses: a review of ideas and approaches

Pterosaurs are an extinct group of Mesozoic flying reptiles, whose fossil record extends from approximately 210 to 66 million years ago. They were integral components of continental and marginal marine ecosystems, yet their diets remain poorly constrained. Numerous dietary hypotheses have been proposed for different pterosaur groups, including insectivory, piscivory, carnivory, durophagy, herbivory/frugivory, filter‐feeding and generalism. These hypotheses, and subsequent interpretations of pterosaur diet, are supported by qualitative (content fossils, associations, ichnology, comparative anatomy) and/or quantitative (functional morphology, stable isotope analysis) evidence. Pterosaur dietary interpretations are scattered throughout the literature with little attention paid to the supporting evidence. Reaching a robustly supported consensus on pterosaur diets is important for understanding their dietary evolution, and their roles in Mesozoic ecosystems. A comprehensive examination of the pterosaur literature identified 314 dietary interpretations (dietary statement plus supporting evidence) from 126 published studies. Multiple alternative diets have been hypothesised for most principal taxonomic pterosaur groups. Some groups exhibit a high degree of consensus, supported by multiple lines of evidence, while others exhibit less consensus. Qualitative evidence supports 87.3% of dietary interpretations, with comparative anatomy most common (62.1% of total). More speciose groups of pterosaur tend to have a greater range of hypothesised diets. Consideration of dietary interpretations within alternative phylogenetic contexts reveals high levels of consensus between equivalent monofenestratan groups, and lower levels of consensus between equivalent non‐monofenestratan groups. Evaluating the possible non‐biological controls on apparent patterns of dietary diversity reveals that numbers of dietary interpretations through time exhibit no correlation with patterns of publication (number of peer‐reviewed publications through time). 73.8% of dietary interpretations were published in the 21st century. Overall, consensus interpretations of pterosaur diets are better accounted for by non‐biological signals, such as the impact of the respective quality of the fossil record of different pterosaur groups on research levels. That many interpretations are based on qualitative, often untestable lines of evidence adds significant noise to the data. More experiment‐led pterosaur dietary research, with greater consideration of pterosaurs as organisms with independent evolutionary histories, will lead to more robust conclusions drawn from repeatable results. This will allow greater understanding of pterosaur dietary diversity, disparity and evolution and facilitate reconstructions of Mesozoic ecosystems.     

The hypersensitive reaction, membrane damage and accumulation of autofluorescent phenolics in lettuce cells challenged by Bremia lactucae

The expression of resistance to Bremia lactucae determined by the resistance genes Dm5/8 and Dm7 in lettuce was examined; incompatibility involved the hypersensitive reaction (HR) which occurred only within penetrated cells at early and late stages of fungal development, respectively. Autofluorescence observed under UV and blue light excitation in cells undergoing the HR was associated with the accumulation of ester-linked syringaldehyde and caffeic acid on plant cell walls. Two phases of phenolic deposition were identified. The first was highly localized around penetration points and occurred during incompatible and compatible interactions. The second and major phase was only activated after the occurrence of irreversible membrane damage in the penetrated cell and was reduced by inhibitors of mRNA synthesis. Fungal structures, primary and secondary vesicles, intercellular hyphae and haustoria also became autofluorescent during incompatible interactions. Changes in the fluorescence due to preformed phenolics located in the plant cell vacuole were found just before plasma membrane damage became irreversible during the HR. In addition to localized deposition of phenolics, increases in the concentrations of the major free phenolic esters identified as dicaffeoyl tartaric and chlorogenic acids also occurred during incompatible interactions. The results suggest that membrane damage in penetrated cells occurs at different rates in resistance controlled by Dm5/8 and Dm7 and indicate an important role for irreversible membrane damage in lettuce as a key signalling event leading to widespread activation of defence responses in surrounding cells.     

Genetic Structure of Rauscher Spleen Focus-Forming Virus

Rauscher spleen focus-forming virus contains functional gag and pol genes and a partially deleted env gene which is structurally related to the env genes of dual tropic murine leukemia viruses.     

Bisnaphthalimidopropyl spermidine induces apoptosis within colon carcinoma cells

Bisnaphthalimido compounds bisintercalate to DNA via the major groove and are potentially potent cancer therapeutics. We incorporated natural polyamines as linkers connecting the two-naphthalimido ring moieties to create a series of novel soluble cytotoxic bisnaphthalimidopropyl polyamines (BNIPPs). Here, we determined the cytotoxicity of bisnaphthalimidopropyl spermidine (BNIPSpd) towards Caco-2 and HT-29 colon adenocarcinoma cells revealing an IC₅₀ value of 0.15 and 1.64 μM after 48 h exposure within Caco-2 and HT-29 cells, respectively. After 4 h, ≥0.5 μM BNIPSpd treatment-induced significant DNA damage. After 24 h exposure a concentration-dependent increase in active caspase-3 expression, chromatin condensation and internucleosomal DNA fragmentation identified apoptosis as the principal manifestation for the cytotoxicity within both cell lines. By 24 h exposure, there was also a significant decline in cellular spermine and spermidine levels. It is concluded that bisnaphthalimidopropyl spermidine (BNIPSpd) toxicity primarily results from apoptosis and that BNISpd has potential to be further developed as an anti-tumour agent.     

Analysis of Leigh Syndrome Mutations in the Yeast SURF1 Homolog Reveals a New Member of the Cytochrome Oxidase Assembly Factor Family

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F²⁴⁹T and Y³⁴⁴D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G¹³⁷E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G¹³⁷E Shy1 mutant phenocopied shy1Δ cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G¹³⁷E Shy1 mutant revealed Coa2, Cox10, and a novel factor designated Coa4. Coa2 and Cox10 are previously characterized CcO assembly factors. Coa4 is a twin CX₉C motif mitochondrial protein localized in the intermembrane space and associated with the inner membrane. Cells lacking Coa4 are depressed in CcO activity but show no impairment in Cox1 maturation or formation of the Shy1-stabilized Cox1 assembly intermediate. To glean insights into the functional role of Coa4 in CcO biogenesis, an unbiased suppressor screen of coa4Δ cells was conducted. Respiratory function of coa4Δ cells was restored by the overexpression of CYC1 encoding cytochrome c. Cyc1 is known to be important at an ill-defined step in the assembly and/or stability of CcO. This new link to Coa4 may begin to further elucidate the role of Cyc1 in CcO biogenesis.Leigh syndrome (LS) is a highly progressive neurological disorder of infancy characterized by necrotizing lesions in the midbrain and brain stem (32). Humans afflicted with LS have compromised oxidative phosphorylation (OXPHOS) function due to mutations in nuclear or mitochondrial genes encoding respiratory chain components or their assembly factors. Although LS infants are born with a normal appearance, neurological lesions develop within months and dysfunction extends to other organs, resulting in a high mortality rate. LS patients typically have mutations affecting complex I or complex IV (cytochrome c oxidase [CcO]) of the OXPHOS pathway (14). Patients with a specific CcO deficiency most often have mutations in the SURF1 gene that encodes a CcO assembly factor (9, 15, 41).SURF1 is not absolutely required for CcO biogenesis in humans, since SURF1-deficient patient fibroblasts retain 10 to 15% of residual CcO activity (32). The yeast homolog of SURF1 is Shy1 (SURF1 homolog in yeast) and has a conserved function in CcO biogenesis (24). Yeast lacking Shy1 retain residual CcO activity, but growth of the mutant strain is compromised on respiratory, nonfermentable carbon sources (4).Insights into the function of SURF1 in human cells have been gleaned through the characterization of stalled CcO assembly intermediates in cells isolated from SURF1 LS patients using blue native (BN) gel electrophoresis. One intermediate, designated S2, which accumulates in SURF1-deficient patient fibroblasts, consists of Cox1 in association with two nuclear CcO subunits, CoxIV and Va (38, 45, 47). A similar stalled assembly intermediate accumulates in CcO-deficient patients with mutations in two other assembly factors, SCO1 and SCO2. These assembly proteins function in the maturation of the mitochondrially encoded Cox2 subunit and the binuclear copper (Cu_A) site within this subunit. In contrast, studies with patient fibroblasts harboring mutations in the genes encoding Cox10 and Cox15 proteins, which are involved in the biosynthesis of the heme a cofactor used exclusively by CcO (at the heme a and heme a₃:Cu_B sites), show only free Cox1 by BN analysis (1, 2). These data suggest that CcO biogenesis commences with the mitochondrial synthesis and maturation of Cox1, while the other two mitochondrially encoded subunits, Cox2 and Cox3, are added at later stages. The absence of the S2 intermediate in cells with mutations in COX10 or COX15 is consistent with the prediction that the S2 assembly intermediate contains Cox1 with at least the heme a center formed.The first major clue to the function of SURF1 came from studies with the bacterium Rhodobacter sphaeroides, in which surf1 mutant cells showed impairment in the formation of the heme a₃:Cu_B bimetallic center within Cox1 (33). Specifically, heme a and Cu_B were observed spectroscopically with surf1 mutant cells, but heme a₃ was not present. The Cu_B site had an altered spectroscopic signature to compensate for the loss of heme a₃, as the two cofactors typically coordinate with each other. This study suggests Surf1 is involved in the maturation of the heme a₃ site in CcO. In lower eukaryotes, impairment of CcO assembly results in proteolytic degradation of the stalled intermediates (16). Thus, it is not possible to isolate the CcO complex in shy1Δ yeast cells to identify any missing cofactors. However, Shy1 was shown to have a key role in formation of the heterobimetallic Cu_B:heme a₃ center in yeast Cox1 (18). Furthermore, it was recently shown that Surf1 in bacteria is a heme-binding protein (10), although these findings have yet to be confirmed in eukaryotes.Additional insights into the function of SURF1/Shy1 came from the isolation of genetic suppressors of shy1Δ respiratory deficiency in yeast (3). Respiratory function can be partially restored in shy1Δ cells by enhancing Cox1 translation through the overexpression of MSS51 (6), a dual-function protein that acts as a COX1 translational activator in addition to binding to the newly synthesized Cox1 polypeptide. Suppression of the shy1Δ respiratory defect is also observed with enhanced expression levels of the two CcO subunits Cox5a and Cox6 corresponding to the human S2-containing subunits CoxIV and Va (15). Overexpression of COA2, a recently identified CcO assembly factor shown to interact with Shy1, can also suppress the shy1Δ respiratory defect (30). Finally, overexpression of the COX10 gene that encodes the hydroxyfarnesyl transferase, which generates heme o as the first step in heme a biosynthesis, can partially restore respiratory function in shy1Δ cells. Although overexpression of COX10 has only very weak suppressor activity, a marked synergistic effect was apparent in the overexpression of both MSS51 and COX10 (29).Shy1 has a secondary function in yeast in the maintenance of the conserved mitochondrial copper storage pool that is used in the copper metallation of Cox1 and Cox2 during CcO biogenesis. Yeast cells lacking Shy1 contain mitochondria with a partially depleted matrix copper storage pool, and the respiratory defect of shy1Δ cells can be partially reversed by growth in the presence of exogenous copper (29). Similarly, liver and muscle samples from patients with SURF1 mutations exhibit a cellular copper deficiency (37). Maintenance of the matrix copper pool is postulated to be linked to active CcO biogenesis in general, as patient tissue with mutations to two other CcO assembly factors, SCO1 and SCO2, result in a cellular copper deficiency as well (22).Human SURF1 and yeast Shy1 are both mitochondrial proteins tethered to the inner membrane (IM) by two transmembrane (TM) helices with a large central domain projecting into the intermembrane space (IMS). Most LS patients with SURF1 mutations have gene deletions or rearrangements. Missense mutations in SURF1 are quite rare, with only a limited number being reported. These mutations tend to be associated with a mild clinical phenotype, and patient survival is prolonged (28). We selected a subset of known missense mutations, two of which lie within the IMS globular domain and a third that maps to the second TM domain. In an attempt to gain further insights into which functional step of SURF1 was compromised by the missense mutations, we engineered and characterized the corresponding mutations in conserved residues of yeast SHY1. In doing so, we have additionally identified a new member of the CcO assembly factor family, Coa4, that may be linked to the role of cytochrome c in CcO assembly. We show that the respiratory defect of cells lacking Coa4 is specifically suppressed by the overexpression of the IMS electron carrier cytochrome c (CYC1). This is the first time CYC1 has been found as a suppressor of a CcO assembly mutant.