Proton NMR assignments and regular backbone structure of bovine pancreatic ribonuclease A in aqueous solution

Proton NMR assignments have been made for 121 of the 124 residues of bovine pancreatic ribonuclease A (RNase A). During the first stage of assignment, COSY and relayed COSY data were used to identify 40 amino acid spin systems belonging to alanine, valine, threonine, isoleucine, and serine residues. Approximately 60 other NH-alpha CH-beta CH systems were also identified but not assigned to specific amino acid type. NOESY data then were used to connect sequentially neighboring spin systems; approximately 475 of the possible 700 resonances in RNase A were assigned in this way. Our assignments agree with those for 20 residues assigned previously [Hahn, U., & Rüterjans, H. (1985) Eur. J. Biochem. 152, 481-491]. Additional NOESY correlations were used to identify regular backbone structure elements in RNase A, which are very similar to those observed in X-ray crystallographic studies [Wlodawer, A., Borkakoti, N., Moss, D. S., & Howlin, B. (1986) Acta Crystallogr. B42, 379-387].     

Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.      

Polyalcohol ligand in Cu and Fe cluster chemistry: Synthesis, structures and magnetic properties of {Cu12} and {Fe8} aggregates

Structurally unique {Cu₁₂} and {Fe₈} cluster complexes were synthesized using 2,2,6,6-tetrakis (hydroxymethyl)cyclohexanol (thcH₅) ligand. The polyalcohol thcH₅ ligand consists of a six membered ring in a chair confirmation and five pendant alcohol arms providing pentadentate-anchoring points for coordination chemistry. A wide range of reaction conditions was explored with transition metal ions in order to isolate large cluster complexes. Obtained {Cu₁₂} and {Fe₈} complexes exhibit unprecedented core topologies where thcH₅ encapsulate and bridge between metal centers and mediate magnetic interactions via the superexchange pathways. Both complexes exhibit dominant intramolecular antiferromagnetic interactions leading to singlet spin ground state.     

Drug-resistant malaria: molecular mechanisms and implications for public health

Resistance to antimalarial drugs has often threatened malaria elimination efforts and historically has led to the short-term resurgence of malaria incidences and deaths. With concentrated malaria eradication efforts currently underway, monitoring drug resistance in clinical settings complemented by in vitro drug susceptibility assays and analysis of resistance markers, becomes critical to the implementation of an effective antimalarial drug policy. Understanding of the factors, which lead to the development and spread of drug resistance, is necessary to design optimal prevention and treatment strategies. This review attempts to summarize the unique factors presented by malarial parasites that lead to the emergence and spread of drug resistance, and gives an overview of known resistance mechanisms to currently used antimalarial drugs.     

Gossypol Increases Expression of the Pro-apoptotic BH3-only Protein NOXA through a Novel Mechanism Involving Phospholipase A2, Cytoplasmic Calcium,and Endoplasmic Reticulum Stress

Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154⁺ stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.     

Contribution of pollen and tapetal characters to the systematics of Triuridaceae

 Pollen and tapetal characters in the mycoheterotrophic monocot family Triuridaceae are here compared with those of their putative relatives, including the lilioid order Pandanales (Pandanaceae, Cyclanthaceae, Velloziaceae and Stemonaceae), with which Triuridaceae have recently been associated following analyses of molecular data. Triuridaceae have small, inaperturate (functionally monoaperturate) pollen grains with the exine reduced to gemmae which have distinctive protruberances or spines. Microsporogenesis is of the successive type. Some genera have a plasmodial tapetum. Orbicules are absent. These characters are compatible with a relationship with Pandanaceae, but a relationship with Alismatales, as suggested by earlier authors, cannot be excluded. Received June 18, 2002; accepted July 22, 2002 Published online: November 22, 2002 Address of the authors: Carol A. Furness (e-mail: c.furness@rbgkew.org.uk), Paula J. Rudall and Alison Eastman, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK.     

Buoyancy adaptations in a swim-bladderless Antarctic fish

The endemic Antarctic teleosts of the suborder Notothenioidei are bottom dwellers. They lack swim bladders, are heavier than seawater, and feed on or near the bottom. The midwaters surrounding the Antarctic continent are productive and underutilized by fishes. There is an evolutionary trend toward pelagism in some notothenioids. We discovered that the largest Antarctic fish, Dissostichus mawsoni, was neutrally buoyant. Attainment of neutral buoyancy was associated with specializations of the skeletal, integumentary, muscular, and digestive systems. The skeleton had a low mineral content and contained considerable cartilage. Scales were also incompletely mineralized. Static lift was obtained from extensive lipid (mostly triglyceride) deposits. A 2–8 mm subcutaneous lipid layer accounted for 4.7% of the body weight. White muscle also contained much lipid–23% on a dry weight basis, or 4.8% of the body weight. Microscopic examination suggested that the liver was active in lipid metabolism, although it was not an organ of buoyancy. Stellate (perisinusoidal) cells with many lipid droplets were a very prominent cytological component of the liver. These specializations made Dissostichus neutrally buoyant and capable of inhabiting the food-rich Antarctic midwaters.