Multiple synthesis by the multipin method as a methodological tool

The multipin method of peptide synthesis is demonstrated as a potent methodological tool, where large numbers of comparative studies can be performed concurrently. Two studies are presented. In each study, the test peptides were simultaneously synthesized, and the products examined by high throughput ion spray mass spectrometry and reverse-phase HPLC. In the first study, comprising 24 experiments, peptides 1 (AELFSTHYLAFKEDYSQ-NH₂) and 2 (LKDFRVYFREGRDQLWKGPG-NH₂) were prepared using Fmoc-Axx/BOP/HOBt/NMM (100: 100: 100: 150 mM ) and Fmoc-Axx/HATU/HOAt/NMM (100: 100: 100: 150 mM ) with 60.90 and 120 min coupling times. The two reagent combinations were found to give comparable results. The second study compared the N-terminal coupling of Fmoc-Asn-OH, Fmoc-Asn(Mbh)-OH, Fmoc-Asn(Mtt)-OH, Fmoc-Asn(Tmob)-OH and Fmoc-Asn(Trt)-OH in the synthesis of seven test peptides: 3 , NVQAAIDYIG-cyclo(Kp); 4 , NTVQAAIDYIG-cyclo(KF); 5 , NRVYVHPFNL; 6 , NRVYVHPFHL: 7 , NEAYVHDAPVRSLN: 8 , NQLVVPSEGLYLIYSQVLFK. 9 , NPNANPNANPNA. A total of 33 experiments were performed. Peptides 3 and 4 were selected to highlight the effect of steric bulk of each Asn derivative on coupling efficiency. Reagent efficiency, as measured by target peptide purity, was as follows: Fmoc-Asn(Tmob)-OH > Fmoc-Asn-OH > Fmoc-Asn(Mtt)-OH = Fmoc-Asn(Trt)-OH > Fmoc-Asn(Mbh)-OH.     

A graphical method for detecting recombination in phylogenetic data sets

Current phylogenetic tree reconstruction methods assume that there is a single underlying tree topology for all sites along the sequence. The presence of mosaic sequences due to recombination violates this assumption and will cause phylogenetic methods to give misleading results due to the imposition of a single tree topology on all sites. The detection of mosaic sequences caused by recombination is therefore an important first step in phylogenetic analysis. A graphical method for the detection of recombination, based on the least squares method of phylogenetic estimation, is presented here. This method locates putative recombination breakpoints by moving a window along the sequence. The performance of the method is assessed by simulation and by its application to a real data set.      

bilbo, a non-LTR retrotransposon of Drosophila subobscura: a clue to the evolution of LINE-like elements in Drosophila

We used the repetitive character of transposable elements to isolate a non-LTR retrotransposon in Drosophila subobscura. bilbo, as we have called it, has homology to TRIM and LOA elements. Sequence analysis showed a 5' untranslated region (UTR), an open reading frame (ORF) with no RNA-binding domains, a downstream ORF that had structural homology to that of the I factor, and, finally, a 3' UTR which ended in several 5-nt repeats. The results of our phylogenetic and structural analyses shed light on the evolution of Drosophila non-LTR retrotransposons and support the hypothesis that an ancestor of these elements was structurally complex.      

International Molybdenum Association (IMOA) life cycle assessment program and perspectives on the LCA harmonization effort

Purpose

In 2001, the International Molybdenum Association (IMOA) initiated their life cycle assessment (LCA) program performing cradle-to-gate life cycle inventories (LCIs) of three molybdenum metallurgical products, followed by LCIs of eight molybdenum chemicals and an update to the metallurgical LCIs. From 2012 to 2014, IMOA participated in a multi-metal industry initiative to harmonize the methodological approach to metal-related LCAs. This paper describes some of IMOA’s conclusions formed from its program and, coupled with its involvement in the multi-metal initiative, provides some lessons learned.

Methods

For this paper, IMOA evaluated the benefits of its LCI program, including its ability to communicate effectively with member companies and stakeholders on the development, use, and application of life cycle data. Likewise, IMOA developed the competence to recognize and provide input on potentially inappropriate use of LCA. IMOA performed a literature review to highlight some of the scientific research using the molybdenum LCI data. IMOA also reviewed the metal industry’s guidance document to provide its perspective on it, including similarities, differences, and substantiation of elements of the four topic areas.

Results and discussion

The metal industry’s guidance document identified four topic areas as essential for alignment with respect to metal-related LCAs: (1) system boundaries, (2) coproduct modelling, (3) life cycle impact assessment (LCIA), and (4) metals recycling modelling. IMOA is largely in agreement with the approaches described in the document. The paper provides examples of how these have been applied to LCAs on Mo-bearing products as well as examples of how some LCA work can benefit from the guidance document.

Conclusions

Having taken part in the harmonization effort, IMOA is poised to educate its member companies and stakeholders about some of the challenging issues encountered in LCA and will continue to lead through active industry participation. IMOA supplies its LCI data via a formal request process which enables open dialogue with stakeholders and LCA practitioners while providing IMOA with insights into how its products fit into the broader lifecycle context and facilitating stakeholders’ awareness of LCA and metals.

     

Variation of physico-chemical parameters along a river transect through the Okavango Delta,Botswana

The Okavango Delta depends on water quantity and quality to sustain its ecosystem services. Whereas many studies have been carried out on its hydrology, few have been done on water quality in the delta. Water pH, electrical conductivity (EC), dissolved oxygen (DO), turbidity, total suspended solids (TSS) and dissolved organic carbon (DOC) were monitored at 10 sites along the Okavango–Boro–Thamalakane–Lake Ngami system almost fortnightly from June 2008 to June 2010. Water quality in the delta was generally good, despite high evapotranspiration rates which would normally produce very saline waters. Electrical conductivity and water temperature increased with distance from Mohembo to Lake Ngami, the former most likely due to evapoconcentration. In contrast, pH, DO, turbidity and TSS decreased with distance from Mohembo to Boro at the lower end of the seasonal floodplain, before increasing again to Lake Ngami. Dissolved oxygen and TSS most likely declined due to biological uptake and particle sedimentation, respectively. Strong and significant relationships were observed between TSS and turbidity and between DOC and EC, indicating that turbidity and EC could be useful proxies for routine estimations of TSS and DOC, respectively, in the delta.     

Macroinvertebrates as bioindicators of water quality in the Mkondoa River,Tanzania, in an agricultural area

The suitability of using macroinvertebrates as bioindicators of stream water quality was tested in the Mkondoa River in an agricultural area at Kilosa, using the rapid bioassessment protocol. The family biotic index (FBI) showed marked variation in water quality along the stream from values ranging from 4.1 to 5.0 in the upstream reaches, indicating good water quality, 5.3 to 5.5 in the mid-reaches and 6.0 to 6.5 in the lower reaches. The water quality index (WQI) indicated that water quality was fair (77 ± 0.98) in the upstream reach of the Mkondoa, marginal (55 ± 0.86) in the midstream reach and poor (33 ± 0.45) in the downstream reach. There were significant relationships between biological oxygen demand and dissolved oxygen and the occurrence of specific taxa, mainly Chironomus and Caenis. Significant changes in macroinvertebrate abundance were mostly related to changes in water quality. As in other parts of the world, macroinvertebrate communities proved to be good biological indicators of water quality and they should be used as bioindicators in long-term monitoring of this river.     

Induction of 32- and 34-kDa stress proteins by sodium arsenite, heavy metals, and thiol-reactive agents

Challenge of human or murine melanoma cells with sodium arsenite, heavy metals (Zn2+, Cu2+ and Cd2+), or thiol-reactive agents (p-chloromercuribenzoate and iodoacetamide) induced the synthesis of four stress proteins with molecular masses of 100, 90, 72 (a doublet), and 32 (human) or 34 (murine) kDa. Enhanced expression of the 32- and 34-kDa polypeptides (p32 and p34) preceded or paralleled the synthesis of the other stress proteins. Hyperthermia, the calcium ionophore A23187, and amino acid analogs (L-azetidine-2-carboxylic acid and L-canavanine) induced the formation of the major stress proteins, but failed to increase synthesis of p32 and p34. Characterization of the dose and time dependence of p32 and p34 synthesis in human (A375) and murine (B16-F10) melanoma cells, respectively, indicated that these proteins were subject to similar regulatory mechanisms. Electrophoretic analysis of stressed cells pulsed with different metabolic precursors revealed that p32 and p34 were radiolabeled with [35S]methionine or 3H-amino acids but not by [3H]mannose or [35S]cysteine. Polyclonal antibodies raised against human p32 cross-reacted with murine p34. These data suggest that p32 and p34 are closely regulated human and murine gene products, respectively, whose synthesis can be modulated by thiol-reactive reagents. Induction of p32 and p34 by these agents, but not by heat shock, suggests that these proteins are a subset of stress-inducible gene products.     

Nephrotoxicity of Penicillium aurantiogriseum,a possible factor in the aetiology of Balkan Endemic Nephropathy

Water-soluble components of a nephrotoxic isolate of Penicillium aurantiogriseum have been fractionated by sequential ion-exchange, size-exclusion gel filtration, reverse-phase silica chromatography and HPLC. Nephrotoxicity in the rat was confined to a size-exclusion fraction approximating to 1500 daltons, which also inhibited DNA synthesis in cultured kidney cells. The more sensitive in vitro assay allowed toxicity to be followed to a sub-fraction from gradient-elution HPLC which in further HPLC resolved into a small group of glycopeptides. Recent Yugoslavian P. aurantiogriseum isolates, from a village in which the idiopathic human disease Balkan Nephropathy is hyperendemic, elicited a similar nephropathology and were acutely cytotoxic, reinforcing a need to regard this novel Penicillium nephrotoxin as a potential factor in human nephropathy.     

Metal contamination in sediments and biota of the Bay of Quinte,Lake Ontario,Canada

The Bay of Quinte receives drainage from several large river systems, including the Moira River which carried sediment from mines into the Bay from the 1880s to the 1960s. We are investigating possible metal contamination of submerged weed beds and marsh biota which may contribute to the low diversity and biomass of macrophyte beds and Typha marshes in the Bay. In 1987, sediment, macrophytes and snails were sampled in wetlands close to the Moira River and at Hay Bay (part of the Bay of Quinte presumably unaffected by mine effluents) located 20 km from the Moira. Some element concentrations in sediment and biota were determined by neutron activation analysis (NAA) including Al, As, Br, Ca, Co, Cl, Cr, Cs, Fe, Hf, K, La, Na, Mg, Sb, Sc, Rb, Ta, Th, Ti, U, V and Zn. Other elements were analysed by acid dissolution and atomic absorption spectrophotometry (AAS) including Ag, As, Cu, Hg, Ni, Pb, and Zn. Levels of As in sediments and plants were higher close to the Moira River, whereas Cu and Ni showed the opposite pattern in sediments. The usefulness of species as bioassays differed: Stagnicola elodes Say accumulated significantly higher levels of Cu (35 vs 18 ppm) and V (l. l vs 0.5 ppm) than Planorbella trivolvis Say collected from the same sites. The macrophyte, Myriophyllum spicatum L. acted as an accumulator of Pb (up to 9.6 ppm), whereas Pb in Vallisneria americana Michx. at the same sites was undetectable.