Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.     

Soil Aggregates and Associated Organic Matter under Conventional Tillage,No-Tillage,and Forest Succession after Three Decades

Impacts of land use on soil organic C (SOC) are of interest relative to SOC sequestration and soil sustainability. The role of aggregate stability in SOC storage under contrasting land uses has been of particular interest relative to conventional tillage (CT) and no-till (NT) agriculture. This study compares soil structure and SOC fractions at the 30-yr-old Horseshoe Bend Agroecosystem Experiment (HSB). This research is unique in comparing NT and CT with adjacent land concurrently undergoing forest succession (FS) and in sampling to depths (15–28 cm) previously not studied at HSB. A soil moving experiment (SME) was also undertaken to monitor 1-yr changes in SOC and aggregation. After 30 years, enhanced aggregate stability under NT compared to CT was limited to a depth of 5 cm, while enhanced aggregate stability under FS compared to CT occurred to a depth of 28 cm and FS exceeded NT from 5–28 cm. Increases in SOC concentrations generally followed the increases in stability, except that no differences in SOC concentration were observed from 15–28 cm despite greater aggregate stability. Land use differences in SOC were explained equally by differences in particulate organic carbon (POC) and in silt-clay associated fine C. Enhanced structural stability of the SME soil was observed under FS and was linked to an increase of 1 Mg SOC ha⁻¹ in 0–5 cm, of which 90% could be attributed to a POC increase. The crushing of macroaggregates in the SME soil also induced a 10% reduction in SOC over 1 yr that occurred under all three land uses from 5–15 cm. The majority of this loss was in the fine C fraction. NT and FS ecosystems had greater aggregation and carbon storage at the soil surface but only FS increased aggregation below the surface, although in the absence of increased carbon storage.     

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.     

Higher plant cell membrane resistance by a single intracellular electrode method

A single intracellular microelectrode technique has been adapted to measure membrane resistance in a higher plant cell. As a direct result of the convenience of this method, which allows relatively long term recordings on a single cell, it has been found that membrane resistance increases for about 30 minutes after cell impalement in Pisum sativum L. cv. Alaska root cortical cells, although cell potential is established at a constant value in less than 2 minutes. It is proposed that these observations imply a regulating feedback loop between electrogenic pump rates and membrane potential.     

Butterfly, bee and forb community composition and cross-taxon incongruence in tallgrass prairie fragments

Pollinators provide an important class of ecological services for crop plants and native species in many ecosystems, including the tallgrass prairie, and their conservation is essential to sustaining prairie remnants. In Iowa these remnants are typically either block-shaped or long, linear strips along transportation routes. In this study we examined differences in the butterfly, bee, and forb community composition in linear and block prairie remnants, determined correlations between species diversity among butterflies, bees and forbs in the 20 prairie remnants sampled, and examined correlations of community similarity among butterflies, bees and forbs. Correspondence analysis showed that distinct communities exist for butterflies and forbs in block versus linear sites and discriminant analysis showed that the bee and forb communities in block and linear sites can be distinguished on the basis of a few species. Diversity of one group was a poor predictor of diversity in another, except for a significant inverse relationship between bees and butterflies. These two pollinator taxa may be responding very differently to microhabitat components within fragmented ecosystems. Our studies show that there need to be differences in conservation strategies for bees and butterflies to maintain both pollinator communities.     

Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg²⁺, DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 °C and 96 °C. In other conditions (absence of Mg²⁺ and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit—probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.     

A SNP haplotype associated with a gene resistant to Xanthomonas axonopodis pv. malvacearum in upland cotton (Gossypium hirsutum L.)

An F_4:5 population of 285 families with each tracing back to a different F₂ plant, derived from a cotton bacterial blight resistant line ‘DeltaOpal’ and a susceptible line ‘DP388’, was artificially inoculated with bacterial blight race 18 (Xanthomonas axonopodis pv. malvacearum) to assay their resistance or susceptibility to the disease. The segregation in the F_4:5 population indicates that the resistance was conditioned by a single dominant gene designated B _12. Simple sequence repeat (SSR) markers identified as putatively linked to the resistance gene by bulked segregant analysis were confirmed on the entire F_4:5 population. Three SSR markers, CIR246, BNL3545 and BNL3644 on chromosome 14, were found closely linked to B ₁₂ . The association between CIR246 and B ₁₂ was validated among 354 plants of 16 diverse varieties. Based on Monsanto SSR/single nucleotide polymorphism (SNP) consensus map, SNP markers closely linked to CIR246 were used to screen ‘DeltaOpal’ and ‘DP388’ for polymorphism. The polymorphic SNP markers were run on the F_4:5 population and the four SNP markers spanning 3.4 cM were found to flank the resistance gene on chromosome 14. The linkage between B ₁₂ and the 4-SNP marker haplotype was validated using 18 elite cotton lines. This 4-SNP marker haplotype can be used for marker assisted selection for bacterial blight resistance breeding programs or for screening germplasm collections for this locus rapidly.     

Crosslinking in viral capsids via tiling theory

A vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. It has been shown in Twarock [2004. A tiling approach to vius capsids assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. This theory is extended here to multi-level tilings in order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.     

Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.     

Formulation of a medical food cocktail for Alzheimer's disease: beneficial effects on cognition and neuropathology in a mouse model of the disease

Background

Dietary supplements have been extensively studied for their beneficial effects on cognition and AD neuropathology. The current study examines the effect of a medical food cocktail consisting of the dietary supplements curcumin, piperine, epigallocatechin gallate, α-lipoic acid, N-acetylcysteine, B vitaminsvitamin C, and folate on cognitive functioning and the AD hallmark features and amyloid-beta (Aβ) in the Tg2576 mouse model of the disease.

Principal Findings

The study found that administering the medical food cocktail for 6 months improved cortical- and hippocampal- dependent learning in the transgenic mice, rendering their performance indistinguishable from non-transgenic controls. Coinciding with this improvement in learning and memory, we found that treatment resulted in decreased soluble Aβ, including Aβ oligomers, previously found to be linked to cognitive functioning.

Conclusion

In conclusion, the current study demonstrates that combination diet consisting of natural dietary supplements improves cognitive functioning while decreasing AD neuropathology and may thus represent a safe, natural treatment for AD.