Immunoaffinity purification of monoclonal antibodies: In search of an elution buffer of general applicability

Summary Of a number of elution buffers tested, 4MgCl2 in 25% ethylene glycol was the most eficient for recovering active monoclonal antibodies from immunoaffinity columns. This solution may be useful as a general purpose eluant for monoclonal antibody purification.     

Ablation of ceramide synthase 2 strongly affects biophysical properties of membranes

Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a ceramide synthase 2 (CerS2)-null mouse, which is unable to synthesize C22-C24 ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.     

Root Rot and Wilt of Kangaroo Paw (Anigozanthos manglesii) Caused by Pythium myriotylum (Drechs.) in Israel

A root rot and wilt disease of Anigozanthos manglesii (Kangaroo Paw) grown in greenhouses in Israel, for exporting as cut flowers to Europe, was characterized. Pythium myriotylum (Drechs.) and Rhizoctonia solani (Kühn) were the prevalent pathogens in diseased plants collected from commercial greenhouses. Fusarium oxysporum, Fusarium spp. and Myrothecium sp. were also isolated, but P. myriotylum or R. solani were not detected in samples from symptomless plants in tissue cultures (Australian origin) or plants at different stages in the nursery; non‐pathogenic F. oxysporum and Fusarium spp. were detected in several samples. In pathogenicity tests carried out in pots, plant mortality occurred 7 days after inoculation with P. myriotylum. In a field experiment carried out in methyl bromide‐fumigated soil, the incidence of dead plants following inoculation with P. myriotylum alone was 22% 10 days after inoculation, increasing to 78% after an additional 25 days. The incidence of dead plants following inoculation with R. solani alone was only 5% and in plants inoculated simultaneously with both pathogens, disease incidence was 88% 35 days after inoculation. Mortality reached 90–100% in plants inoculated with P. myriotylum, either singly or combined with R. solani 60 days after inoculation, whereas in plants inoculated with R. solani it was 5%. The maximum mortality in plants inoculated with R. solani was 25%, 76 days after inoculation. These results clearly demonstrate that P. myriotylum was the dominant pathogen in the root rot and wilt of A. manglesii.     

Assessment of anadromous salmon resources in the diet of the Alexander Archipelago wolf using stable isotope analysis

The Alexander Archipelago wolf (Canis lupus ligoni) is unique to southeast Alaska, occurring on islands south of Frederick Sound and along the mainland between Dixon Entrance and Yakutat Bay. Sitka black-tailed deer (Odocoileus hemionus sitkensis) are an important prey species for wolves across the southern part of the region. Spawning salmon (Onchorynchus sp.) are seasonally available but their presence in wolf diets has not previously been quantified. We examined the range of bone collagen δ¹³C and δ¹⁵N values for wolves throughout southeast (n = 163) and interior (n = 50) Alaska and used a dual-isotope mixing model to determine the relative contribution of salmon-derived marine protein in the diet. Southeast Alaska wolves consumed significantly more salmon (mean ± SE: 18.3 ± 1.2%) than did wolves from interior Alaska (9.1 ± 0.6%, P<0.001). Wolves on the southeast Alaska mainland appeared to have higher marine isotopic signatures than island wolves, although this difference was not significant. Variation among individual wolf diets was higher for southeast than for interior Alaska wolves, and variation was highest in coastal mainland wolf diets (P<0.001). Marine resources may augment the diet of southeast Alaska wolves during seasonal or annual fluctuations in the availability of deer, particularly in those areas on the mainland where densities of terrestrial ungulates are relatively low. Received: 1 July 1998 / Accepted: 23 February 1999     

Charge properties of human pituitary and amniotic fluid prolactins.

The apparent isoelectric points (pI) in isoelectric focusing (IF) of human pituitary and amniotic fluid prolactin (hPRL), both non-iodinated and iodinated, were determined. Unresolved mixtures of pituitary hPRL isohormones E and F, and of at least five isohormones found in amniotic fluid, and plasma hPRL exhibit an average pI value of 6.5 - 6.7. Transient state pH values observed or previously reported for hPRL components range from pH 5.9 to 6.8 after correction to standard conditions. At pH 8.1, the major isohormone, hPRL-F, carriers a charge of 2.2 net protons per molecule. The net charge differences among isohormones E, F and G are compatible with acquisition or loss of single charged groups per 20,000 molecular weight. This net charge is similar to that of the least prolactin-bioactive major isohormone of human growth hormone (hGH-B), while the hGH with a bioactivity comparable to that of hPRL exhibits a net charge of 3.4 valence units. The "large" isohormones J and H increased net charges, by a factor of 2-3, in direct proportion to their size increments.     

Nickel promoted C-H, C-C and C-O bond activation in solution

Reaction of NiI₂ with the PCP-ligand {1-Et-2,6-(CH₂PⁱPr₂)₂-C₆H₃} (1) results in selective activation of the strong sp²-sp³ aryl-ethyl bond to afford the aryl-nickel complex [Ni{2,6-(CH₂PⁱPr₂)₂-C₆H₃}I] (2), whereas reaction of NiI₂ with {1,3,5-(CH₃)₃-2,6-(CH₂PⁱPr₂)₂-C₆H} (4) leads to the formation of the benzylic complex [Ni{1-CH₂-2,6-(CH₂PⁱPr₂)₂-3,5-(CH₃)₂-C₆H}I] (5) by selective C-H bond activation. Thermolysis of 5 results in formation of [Ni{2,6-(CH₂PⁱPr₂)₂-3,5-(CH₃)₂-C₆H}I] (6) by activation of the sp²-sp³ C-C bond. The identity of the new 16-electron complexes 2 and 6 was confirmed by reaction of NiI₂ with {1,3-(CH₂PⁱPr₂)₂-C₆H₄} (3) and {1,3-(CH₃)₂-4,6-(CH₂PⁱPr₂)₂-C₆H₂} (7), respectively, lacking the aryl-alkyl groups between the “phosphines arms” (alkyl=ethyl, methyl). Complexes 2 and 5 have been fully characterized by X-ray analysis. Nickel-based activation of an unstrained C-O single bond was observed as well. Reaction of the aryl-methoxy bisphosphine {1-OMe-2,6-(CH₂PⁱPr₂)₂-C₆H₃} (8) with NiI₂ results in the formation of the phenoxy complex [Ni{1-O-2,6-(CH₂PⁱPr₂)₂-C₆H₃}I] (9) by selective sp³-sp³ C-O bond activation.     

sp C-H and sp C-H agostic ruthenium complexes: a combined experimental and theoretical study

Halide abstraction from the 18 electron Ru(II) complex RuCl(CO)₂[2,6-(CH₂P^tBu₂)₂C₆H₃] (2) with AgPF₆ results in the exclusive formation of the cationic complex {Ru(CO)₂[2,6-(CH₂P^tBu₂)₂C₆H₃]}⁺PF₆⁻ (3). The molecular structures of 2 and 3 were determined by complete single-crystal diffraction studies. X-ray crystallographic analysis of 3 reveals that the “open” coordination site is occupied by an agostic interaction between the metal center and an sp³ C-H bond of a tert-butyl substituent. DFT gas phase calculations (B97-1/SDD) show the necessity of two sterically demanding tert-butyl substituents on one P donor atom for the agostic interaction to occur. The reaction of 3 with H₂ results in the quantitative conversion to {Ru(H)(CO)₂[2,6-(CH₂P^tBu₂)₂C₆H₄]}⁺PF₆⁻ (4) where the aromatic C_ipso-H bond is η²-coordinated to the metal center. Treatment of the agostic complex 4 with Et₃N results in the formation of the neutral complex Ru(H)(CO)₂[2,6-(CH₂P^tBu₂)₂C₆H₃] (5). The mechanistic details of 3 + H₂ → 4 were investigated by DFT calculations at the B97-1/SDB-cc-pVDZ//B97-1/SDD level of theory.