Timing and climate forcing of volcanic eruptions for the past 2,500 years.

Volcanic eruptions contribute to climate variability, but quantifying these contributions has been limited by inconsistencies in the timing of atmospheric volcanic aerosol loading determined from ice cores and subsequent cooling from climate proxies such as tree rings. Here we resolve these inconsistencies and show that large eruptions in the tropics and high latitudes were primary drivers of interannual-to-decadal temperature variability in the Northern Hemisphere during the past 2,500 years. Our results are based on new records of atmospheric aerosol loading developed from high-resolution, multi-parameter measurements from an array of Greenland and Antarctic ice cores as well as distinctive age markers to constrain chronologies. Overall, cooling was proportional to the magnitude of volcanic forcing and persisted for up to ten years after some of the largest eruptive episodes. Our revised timescale more firmly implicates volcanic eruptions as catalysts in the major sixth-century pandemics, famines, and socioeconomic disruptions in Eurasia and Mesoamerica while allowing multi-millennium quantification of climate response to volcanic forcing.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes.

The DNA sequence of 91.4 kilobases of the Escherichia coli K-12 genome, spanning the region between rrnC at 84.5 minutes and rrnA at 86.5 minutes on the genetic map (85 to 87 percent on the physical map), is described. Analysis of this sequence identified 82 potential coding regions (open reading frames) covering 84 percent of the sequenced interval. The arrangement of these open reading frames, together with the consensus promoter sequences and terminator-like sequences found by computer searches, made it possible to assign them to proposed transcriptional units. More than half the open reading frames correlated with known genes or functions suggested by similarity to other sequences. Those remaining encode still unidentified proteins. The sequenced region also contains several RNA genes and two types of repeated sequence elements were found. Intergenic regions include three "gray holes," 0.6 to 0.8 kilobases, with no recognizable functions.

The complete genome sequence of Escherichia coli K-12.

The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Novel phosphotransferase-encoding genes revealed by analysis of the Escherichia coli genome: a chimeric gene encoding an Enzyme I homologue that possesses a putative sensory transduction domain.

Two genes (ptsI and ptsA) that encode homologues of the energy coupling Enzyme I of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system (PTS) have previously been identified on the Escherichia coli chromosome. We here report the presence of a third E. coli gene, designated ptsP, that encodes an Enzyme I homologue, here designated Enzyme INtr. Enzyme INtr possesses an N-terminal domain homologous to the N-terminal domains of NifA proteins [(127 amino acids (aa)] joined via two tandem flexible linkers to the C-terminal Enzyme I-like domain (578 aa). Structural features of the putative ptsP operon, including transcriptional regulatory signals, are characterized. We suggest that Enzyme INtr functions in transcriptional regulation of nitrogen-related operons together with previously described PTS proteins encoded within the rpoN operon. It may thereby provide a link between carbon and nitrogen assimilatory pathways.

Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli.

Sequences of four new heat-shock (HS) genes of Escherichia coli organized into two operons were determined. The operon at 83 min specifies two proteins of 15.8 kDa (HslT) and 16.1 kDa (HslS), which are identical to IbpA and IbpB, respectively. Expression of mRNA from a sigma 32-dependent promoter of the hslTS/ibpAB operon is stimulated 30-75-fold upon temperature upshift. The transcription start point (tsp) is located at a G, 96 bp upstream from the AUG start codon of hslT/ibpA. The deduced amino acid sequences of HslT/IbpA and HslS/IbpB are 48% identical to each other and were found to be remotely related to the chloroplast low-molecular-weight HS protein, which is highly conserved among plants. The second hs operon is much less actively stimulated by temperature upshift, although it has a hs promoter that perfectly matches the consensus of promoters recognized by sigma 32. Located at 88.9 min, the hslVU operon specifies proteins of 19.1 kDa (HslV) and 49.6 kDa (HslU). Multiple tsp were found in this operon. HslV is remotely related to the eukaryotic proteasome proteins, and HslU is very similar to a Pasteurella haemolytica protein of unknown function. Both HslU and the P. haemolytica protein share a ATP/GTP-binding motif near their N-termini. The two operons described here are transcribed counterclockwise on the standard genetic map.

Subdivision of the Escherichia coli K-12 genome for sequencing: manipulation and DNA sequence of transposable elements introducing unique restriction sites.

A transposon-based method of introducing unique restriction sites was used for subdivision of the Escherichia coli genome into a contiguous series of large non-overlapping segments spanning 2.5Mb. The segments, sizes ranging from 150 to 250kb, were isolated from the chromosome using the inserted restriction sites and shotgun cloned into an M13 vector for DNA sequencing. These shotgun sizes proved easily manageable, allowing the genomic sequence of E. coli to be completed more efficiently and rapidly than was possible by previously available methods. The 9bp duplication generated during transposition was used as a tag for accurate splicing of the segments; no further sequence redundancy at the junction sites was needed. The system is applicable to larger genomes even if they are not already well-characterized. We present the technology for segment sequencing, results of applying this method to E. coli, and the sequences of the transposon cassettes.

Thermal injury during pregnancy.

Nineteen pregnant women with burn areas ranging from 6 to 92% of the total body surface area were treated at the US Army Institute of Surgical Research between 1951 and 1974. A review of these cases led to the following observations and conclusions: 1) Pregnancy does not alter the maternal outcome after thermal injury. 2) Maternal survival is usually accompanied by fetal survival. 3) If the gravida's injury is lethal, the pregnancy will usually terminate spontaneously prior to her death. 4) Obstetric intervention is indicated only in the gravely ill woman whose complications (hypotension, hypoxia, or sepsis) jeopardize the life of the fetus. 5) A better understanding of the complications of major burns and the care available in modern burn units should improve the prognosis for burned pregnant patients.

Design and operation of a fast electromagnetic inductive massive gas injection valve for NSTX-U.

Results from the operation of an electromagnetic valve, that does not incorporate ferromagnetic materials, are presented. Image currents induced on a conducting disc placed near a pancake solenoid cause it to move away from the solenoid and open the vacuum seal. A new and important design feature is the use of Lip Seals for the sliding piston. The pressure rise in the test chamber is measured directly using a fast time response Baratron gauge. The valve injects over 200 Torr l of nitrogen in less than 3 ms, which remains unchanged at moderate magnetic fields.

Retroregulation of the bacteriophage lambda int gene: limited secondary degradation of the RNase III-processed transcript.

Expression of the int gene of bacteriophage lambda from two promoters, pI and pL, is differentially regulated through RNA processing. Efficient Int protein synthesis from the pL RNA is inhibited by the action of sib, a cis-acting retroregulator downstream from the int gene. We have used mapping procedures with nuclease S1 to study the pL transcripts produced in vivo after phage lambda infection. We have found an RNase III-dependent processing site within the Int coding sequence, 387 nucleotides upstream from the site of the primary cleavage by RNase III at Sib. This secondary processing site is located at the most stable region of secondary structure in the sib int region, as predicted by computer analysis. We suggest that RNase III cleavage at the Sib site allows processive exonucleolytic degradation of the RNA to proceed to a region of secondary structure within the Int coding sequence, which protects the upstream region of the transcript from further degradation.

Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities.

The single disulfide bond in potato Inhibitor I protomers (Mr = 8,000) was reduced and alkylated in the absence of denaturants to the carboxamidomethyl cysteine derivative. Two half-cystines per mol of inhibitor protomer were modified as determined by (a) loss of two sulfhydryl groups per reduced protomer, (b) incorporation of 2 mol of [14C]iodoacetamide per protomer, and (c) amino acid analyses. The alkylated Inhibitor I retained its oligomeric structure (Mr = 40,000) and fully retained its immunological cross reactivity with anti-Inhibitor I serum. Furthermore, the inhibitory properties of modified and unmodified Inhibitor I toward chymotrypsin were identical. The stoichiometric complex between modified Inhibitor I and chymotrypsin was stable at pH 8.0 for over 72 h. The modification of the disulfide bond introduced a lability to both heat and proteolytic enzymes. Ultraviolet difference spectra and near ultraviolet circular dichroism spectra at pH 8.0, between modified and unmodified Inhibitor I, revealed only minor absorption changes due to modification. However, in circular dichroism spectra at pH 2.0, the modified inhibitor exhibited a significant loss of absorption in the region between 270 and 280 nm that was present in the unmodified inhibitor. The cumulative results of this study indicate that the single disulfide bond in Inhibitor I, which forms a rather large disulfide loop between residues 5 and 51, is not important at neutral pH for maintaining major structural conformations which affect immunological or inhibitory activities, but the disulfide bond does impose restraints on the protein which stabilize it toward thermal denaturation and proteolysis. The susceptibility of the reduced inhibitor to plant sulfhydryl enzymes may have some importance in in vivo degradation.

Relationships among "ancient araliads" and their significance for the systematics of Apiales.

The relationship between the angiosperm families Apiaceae and Araliaceae (order Apiales) has been difficult to resolve, due in large part to problems associated with taxa characterized by a mixture of features typical of both families. Among such confounding groups are the araliads Delarbrea, Pseudosciadium, Myodocarpus, Mackinlaya, and Apiopetalum and many members of Apiaceae subfamily Hydrocotyloideae. Traditional systems have often envisioned these taxa as phyletic intermediates or bridges between the two families. To reevaluate the phylogenetic position of the "intermediate" araliad genera, molecular data were collected from nuclear (rDNA ITS) and plastid (matK) sequences from a complete or near-complete sampling of species in each genus. When analyzed with samples representing the other major clades now recognized within Apiales, results confirm and expand the findings of previously published studies. The five araliad "intermediates" are placed within two well-supported clades clearly segregated from the "core" groups of both Apiaceae and Araliaceae. These segregate clades closely parallel traditional definitions of the araliad tribes Myodocarpeae (Delarbrea, Pseudosciadium, and Myodocarpus) and Mackinlayeae (Mackinlaya and Apiopetalum), and relationships among the species within these clades are largely supported by morphological and anatomical data. Based on these results, Myodocarpeae and Mackinlayeae may best be treated as distinct families. This approach would render four monophyletic groups within Apiales, to which a fifth, Pittosporaceae, cannot at present be excluded. Sampling of taxa from Hydrocotyloideae remains preliminary, but results confirm previous studies indicating the polyphyly of this subfamily: hydrocotyloid taxa may be found in no fewer than three major clades in Apiales.

Clarification of the relationship beteen Apiaceae and Araliaceae based on matK and rbcL sequence data.

Apiaceae and Araliaceae (Apiales) represent a particularly troublesome example of the difficulty in understanding evolutionary relationships between tropical-temperate family pairs. Previous studies based on rbcL sequence data provided insights at higher levels, but were unable to resolve fully the family-pair relationship. In this study, sequence data from a more rapidly evolving gene, matK, was employed to provide greater resolution. In Apiales, matK sequences evolve an average of about two times faster than rbcL sequences. Results of phylogenetic analysis of matK sequences were first compared to those obtained previously from rbcL data; the two data sets were then combined and analyzed together. Molecular analyses confirm the polyphyly of apiaceous subfamily Hydrocotyloideae and suggest that some members of this subfamily are more closely related to Araliaceae than to other Apiaceae. The remainder of Apiaceae forms a monophyletic group with well-defined subclades corresponding to subfamilies Apioideae and Saniculoideae. Both the matK and the combined rbcL-matK analyses suggest that most Araliaceae form a monophyletic group, including all araliads sampled except Delarbrea and Mackinlaya. The unusual combination of morphological characters found in these two genera and the distribution of matK and rbcL indels suggest that these taxa may be the remnants of an ancient group of pro-araliads that gave rise to both Apiaceae and Araliaceae. Molecular data indicate that the evolutionary history of the two families is more complex than simple derivation of Apiaceae from within Araliaceae. Rather, the present study suggests that there are two well-defined "families," both of which may have been derived from a lineage (or lineages) or pro-araliads that may still have extant taxa.

Major lineages within Apiaceae subfamily Apioideae: a comparison of chloroplast restriction site and DNA sequence data.

Traditional sources of taxonomic characters in the large and taxonomically complex subfamily Apioideae (Apiaceae) have been confounding and no classification system of the subfamily has been widely accepted. A restriction site analysis of the chloroplast genome from 78 representatives of Apioideae and related groups provided a data matrix of 990 variable characters (750 of which were potentially parsimony-informative). A comparison of these data to that of three recent DNA sequencing studies of Apioideae (based on ITS, rpoCl intron, and matK sequences) shows that the restriction site analysis provides 2.6-3.6 times more variable characters for a comparable group of taxa. Moreover, levels of divergence appear to be well suited to studies at the subfamilial and tribal levels of Apiaceae. Cladistic and phenetic analyses of the restriction site data yielded trees that are visually congruent to those derived from the other recent molecular studies. On the basis of these comparisons, six lineages and one paraphyletic grade are provisionally recognized as informal groups. These groups can serve as the starting point for future, more intensive studies of the subfamily.