The interacting effects of elevated atmospheric CO<Subscript>2</Subscript> concentration,drought and leaf-to-air vapour pressure deficit on ecosystem isoprene fluxes

Isoprene is the most abundant biogenic hydrocarbon released from vegetation and it plays a major role in tropospheric chemistry. Because of its link to climate change, there is interest in understanding the relationship between CO₂, water availability and isoprene emission. We explored the effect of atmospheric elevated CO₂ concentration and its interaction with vapour pressure deficit (VPD) and water stress, on gross isoprene production (GIP) and net ecosystem exchange of CO₂ (NEE) in two Populus deltoides plantations grown at ambient and elevated atmospheric CO₂ concentration in the Biosphere 2 Laboratory facility. Although GIP and NEE showed a similar response to light and temperature, their responses to CO₂ and VPD were opposite; NEE was stimulated by elevated CO₂ and depressed by high VPD, while GIP was inhibited by elevated CO₂ and stimulated by high VPD. The difference in response between isoprene production and photosynthesis was also evident during water stress. GIP was stimulated in the short term and declined only when the stress was severe, whereas NEE started to decrease from the beginning of the experiment. This contrasting response led the carbon lost as isoprene in both the ambient and the elevated CO₂ treatments to increase as water stress progressed. Our results suggest that water limitation can override the inhibitory effect of elevated CO₂ leading to increased global isoprene emissions in a climate change scenario with warmer and drier climate.     

Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions

Timed embryo transfer (TET) using in vitro produced (IVP) embryos without estrus detection can be used to reduce adverse effects of heat stress on fertility. One limitation is the poor survival of IVP embryos after cryopreservation. Objectives of this study were to confirm beneficial effects of TET on pregnancy rate during heat stress as compared to timed artificial insemination (TAI), and to determine if cryopreservation by vitrification could improve survival of IVP embryos transferred to dairy cattle under heat stress conditions. For vitrified embryos (TET-V), a three-step pre-equilibration procedure was used to vitrify excellent and good quality Day 7 IVP Holstein blastocysts. For fresh IVP embryos (TET-F), Holstein oocytes were matured and fertilized; resultant embryos were cultured in modified KSOM for 7 days using the same method as for production of vitrified embryos. Excellent and good quality blastocysts on Day 7 were transported to the cooperating dairy in a portable incubator. Nonpregnant, lactating Holsteins (n = 155) were treated with GnRH (100 microg, i.m., Day 0), followed 7 days later by prostaglandin F2alpha (PGF2alpha, 25 mg, i.m.) and GnRH (100 microg) on Day 9. Cows in the TAI treatment (n = 68) were inseminated the next day (Day 10) with semen from a single bull that also was used to produce embryos. Cows in the other treatments (n = 33 for TET-F; n = 54 for TET-V) received an embryo on Day 17 (i.e. Day 7 after anticipated ovulation and Day 8 after second GnRH treatment). The proportion of cows that responded to synchronization based on plasma progesterone concentrations on Day 10 and Day 17 was 67.7%. Pregnancy rate for all cows on Day 45 was higher (P < 0.05) in the TET-F treatment than for the TAI and TET-V treatments (19.0 +/- 5.0,6.2 +/- 3.6, and 6.5 +/- 4.1%). For cows responding to synchronization, pregnancy rate was also higher (P < 0.05) for TET-F than for other treatments (26.7 +/- 6.4, 5.0 +/- 4.3, and 7.4 +/- 4.7%). In the TET-F treatment group, cows producing more milk had lower (P < 0.05) pregnancy rates than cows producing less milk. In conclusion, ET of fresh IVP embryos can improve pregnancy rate under heat stress conditions, but pregnancy rate following transfer of vitrified embryos was no better than that following TAI.     

Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture

Cryopreservation causes a significant proportion of bovine oocytes to undergo degeneration during subsequent culture. We investigated the degeneration mechanism of cryopreserved oocytes. In vitro matured bovine oocytes were vitrified by the open-pulled straw (OPS) method. In each replicate, a group of oocytes were randomly taken after warming to determine oocyte survival by both morphological evaluation and propidium iodide vital staining. The remainders were evaluated by morphological criterion. Morphologically intact oocytes were co-incubated with frozen-thawed spermatozoa for subsequent development. In situ examination of DNA breaks in oocytes and embryos was conducted using a Fluorescein-FragEL DNA fragmentation detection kit. A caspase-3 detection kit was used to detect caspase-3 activity in oocytes and embryos. Most of the oocytes survived cooling and warming processes as assessed by both morphological evaluation and vital stain. During subsequent culture, some degenerating oocytes displayed observable apoptotic morphology, such as cytoplasmic condensation, cytoplasmic fragmentation, and formation of apoptotic bodies. Biochemical markers of apoptosis, such as apoptotic DNA fragmentation and activation of caspases, were detected not only in oocytes having typical apoptotic morphology, but also in oocytes without observable apoptotic morphology. In embryos, positive signals for both biochemical markers were detected in blastomeres. This experiment suggests that cryopreserved bovine oocytes degenerate via apoptosis during subsequent culture.     

Cell allocation in bovine embryos cultured in two media under two oxygen concentrations

Blastocyst development, total cell number and allocation to inner cell mass (ICM) and trophectoderm (TE) lineages was compared among day 9 hatched blastocysts from four culture treatments in a two-factor design. Two modified commercial media (KSOM and SOF) were used in atmospheres with two oxygen concentrations (5% and 20% O2). No significant effect of medium on development was found, but 20% O2 increased hatching (p < 0.05). There were more cells in hatched blastocysts cultured in KSOM than in SOF (181 vs 136, respectively; p < 0.0001); however, ICM/total cell ratio was not affected by medium. There was a trend suggesting that the proportion of cells allocated to ICM was lower in hatched blastocysts cultured under 5% O2 compared with 20% O2 (0.323 vs 0.380, respectively; p < 0.1). No significant interactions between medium type and oxygen concentration were found. These results indicate that culture components used in this study may affect cell proliferation without altering cell allocation, and that oxygen concentration may play a role in allocation of cells to ICM and TE lineages.     

Schizosaccharomyces pombe pfh1+ encodes an essential 5' to 3' DNA helicase that is a member of the PIF1 subfamily of DNA helicases

The Saccharomyces cerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans. S. cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA. Here we describe the isolation and characterization of pfh1+, a Schizosaccharomyces pombe gene that encodes a Pif1-like protein. Pfh1p was the only S. pombe protein with high identity to Saccharomyces Pif1p. Unlike the two S. cerevisiae Pif1 subfamily proteins, the S. pombe Pfh1p was essential. Like Saccharomyces Pif1p, a truncated form of the S. pombe protein had 5' to 3' DNA helicase activity. Point mutations in an invariant lysine residue in the ATP binding pocket of Pfh1p had the same phenotype as deleting pfh1+, demonstrating that the ATPase/helicase activity of Pfh1p was essential. Although mutant spores depleted for Pfh1p proceeded through S phase, they arrested with a terminal cellular phenotype consistent with a postinitiation defect in DNA replication. Telomeric DNA was modestly shortened in the absence of Pfh1p. However, genetic analysis demonstrated that maintenance of telomeric DNA was not the sole essential function of S. pombe Pfh1p.     

Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees (Apis mellifera)

Four antimicrobial peptides were purified from Royal Jelly of honeybees, by using reverse phase-HPLC and sequenced by using Q-Tof-MS/MS: PFKLSLHL-NH(2) (Jelleine-I), TPFKLSLHL-NH(2) (Jelleine-II), EPFKLSLHL-NH(2) (Jelleine-III), and TPFKLSLH-NH(2) (Jelleine-IV). The peptides were synthesized on-solid phase, purified and submitted to different biological assays: antimicrobial activity, mast cell degranulating activity and hemolysis. The Jelleines-I-III presented exclusively antimicrobial activities against yeast, Gram+ and Gram- bacteria; meanwhile, Jelleine-IV was not active in none of the assays performed. These peptides do not present any similarity with the other antimicrobial peptides from the honeybees; they are produced constitutively by the workers and secreted into Royal Jelly.     

The V lambda J lambda repertoire in human fetal spleen: evidence for positive selection and extensive receptor editing

VlambdaJlambda rearrangements obtained from genomic DNA of individual IgM(+) B cells from human fetal spleen were analyzed. A nonrandom pattern of lambda gene rearrangements that differed from the adult Vlambda repertoire was found. The Vlambda distal genes 8A and 4B were absent from the nonproductive fetal repertoire, whereas 2E and 3L were overrepresented and 1B was underrepresented in the productive fetal repertoire. Positive selection of the Vlambda gene, 2E, along with Vlambda rearrangements employing homologous VlambdaJlambda joins were observed in the fetal, but not in the adult Vlambda repertoire. Overrepresentation of Jlambda distal cluster C genes rearranging to the Vlambda distal J segment, Jlambda7, in both productive and nonproductive fetal repertoires suggested that receptor editing/replacement was more active in the fetus than in adults. Numerous identical VlambdaJlambda junctions were observed in both the productive and nonproductive repertoire of the fetus and adult, but were significantly more frequent in the productive repertoire of the fetus, suggesting expansion of B cells expressing particular lambda-light chains in both stages of development, with more profound expansion in the fetal repertoire. Notably, B cells expressing identical lambda-light chains expressed diverse heavy chains. These data demonstrate that three mechanisms strongly influence the shaping of the human fetal lambda-chain repertoire that are less evident in the adult: positive selection, receptor editing, and expansion of B cells expressing specific lambda-light chains. These events imply that the expressed fetal repertoire is shaped by exposure to self Ags.     

PHOTOSYNTHETIC CHARACTERISTICS OF C3-C4 INTERMEDIATE FLAVERIA FLORIDANA (ASTERACEAE) IN NATURAL HABITATS: EVIDENCE OF ADVANTAGES TO C3-C4 PHOTOSYNTHESIS AT HIGH LEAF TEMPERATURES

Measurements of leaf gas exchange were conducted in situ for the C₃-C₄ intermediate plant Flaveria floridana. Leaves exhibited measurable CO₂ assimilation at atmospheric CO₂ concentrations as low as 20 μmol/mol. This result demonstrates that the low CO₂ compensation points observed in past studies of greenhouse-grown C₃-C₄ intermediate plants also exist in plants growing in their natural habitat. Photosynthesis rates in F. floridana were near their maximum at intercellular CO₂ concentrations as low as 112 μmol/mol. The existence of near-maximum photosynthesis rates at such low intercellular CO₂ concentrations is interpreted as evidence for the existence of a CO₂-concentrating mechanism in F. floridana. Such a mechanism would also explain the observed lack of response in photosynthesis rates to reductions in stomatal conductance and intercellular CO₂ concentration as the leaf-to-air water vapor concentration gradient is increased. Photosynthetic rates were relatively high at leaf temperatures between 35 and 40 C, compared to most C₃ plants. At midday during May, when leaf temperatures were between 35 and 42 C, F. floridana leaves exhibited photosynthesis rates that were four times higher than a sympatric C₃ species (Eustoma exaltatum) of similar growth form and ecological habit. The high photosynthesis rates at high leaf temperatures in F. floridana were not due to higher leaf nitrogen contents, but rather to its reduced rate of photorespiration. These results confirm that C₃-C₄ intermediate photosynthesis can provide plants with an advantage at high leaf temperatures, compared to C₃ plants.     

Presentation Overrides Specificity: Probing the Plasticity of Alphaviral Proteolytic Activity through Mutational Analysis

Semliki Forest virus (genus Alphavirus) is an important model for studying regulated nonstructural (ns) polyprotein processing. In this study, we evaluated the strictness of the previously outlined cleavage rules, accounting for the timing and outcome of each of three cleavages within the ns polyprotein P1234, and assessed the significance of residues P6 to P4 within the cleavage sites using an alanine scanning approach. The processing of the 1/2 and 3/4 sites was most strongly affected following changes in residues P5 and P4, respectively. However, none of the mutations had a detectable effect on the processing of the 2/3 site. An analysis of recombinant viruses bearing combinations of mutations in cleavage sites revealed tolerance toward the cooccurrence of native and mutated cleavage sites within the same polyprotein, suggesting a remarkable plasticity of the protease recognition pocket. Even in a virus in which all of the cleavage sequences were replaced with alanines in the P6, P5, and P4 positions, the processing pattern was largely preserved, without leading to reversion of cleavage site mutations. Instead, the emergence of second-site mutations was identified, among which Q706R/L in nsP2 was confirmed to be associated with the recognition of the P4 position within the modified cleavage sites. Our results imply that the spatial arrangement of the viral replication complex inherently contributes to scissile-site presentation for the protease, alleviating stringent sequence recognition requirements yet ensuring the precision and the correct order of processing events. Obtaining a proper understanding of the consequences of cleavage site manipulations may provide new tools for taming alphaviruses.