Structural basis for binding of cyclic 2-oxoglutarate analogues to factor-inhibiting hypoxia-inducible factor

Aromatic analogues of the 2-oxoglutarate co-substrate of the hypoxia-inducible factor hydroxylases are shown to bind at the active site iron: Pyridine-2,4-dicarboxylate binds as anticipated with a single molecule chelating the iron in a bidentate manner. The binding mode of a hydroxamic acid analogue, at least in the crystalline state, is unusual because two molecules of the inhibitor are observed at the active site and partial displacement of the iron binding aspartyl residue was observed.     

Hemoglobin E: a common hemoglobinopathy among children of Southeast Asian origin.

With the recent immigration of Southeast Asians to Canada, hemoglobin E has become a frequent diagnosis. The clinical and hematologic findings in 42 children (mean age 4.3 years) with hemoglobin E are presented. There were 33 heterozygotes (having hemoglobin E trait), 6 homozygotes (having hemoglobin EE) and 3 double heterozygotes (having hemoglobin E-beta-thalassemia). The heterozygotes had low-normal hemoglobin levels and mean corpuscular volumes; coexisting iron deficiency, present in 62% of these children, resulted in substantially lower hemoglobin levels, very low mean corpuscular volumes and lower than expected levels of hemoglobin E on electrophoresis. The children with hemoglobin EE were only slightly anemic, but those with hemoglobin E-beta-thalassemia had severe anemia and required long-term transfusion therapy. Nutritional factors and parasitic infestations were the main causes of iron depletion, which was common, particularly in children less than 2 years old (87%). Physicians of patients of Southeast Asian origin should be aware of the clinical and hematologic presentation of these hemoglobinopathies.     

Dual role of the cysteine-string domain in membrane binding and palmitoylation-dependent sorting of the molecular chaperone cysteine-string protein

S-palmitoylation occurs on intracellular membranes and, therefore, membrane anchoring of proteins must precede palmitate transfer. However, a number of palmitoylated proteins lack any obvious membrane targeting motifs and it is unclear how this class of proteins become membrane associated before palmitoylation. Cysteine-string protein (CSP), which is extensively palmitoylated on a string of 14 cysteine residues, is an example of such a protein. In this study, we have investigated the mechanisms that govern initial membrane targeting, palmitoylation, and membrane trafficking of CSP. We identified a hydrophobic 31 amino acid domain, which includes the cysteine-string, as a membrane-targeting motif that associates predominantly with endoplasmic reticulum (ER) membranes. Cysteine residues in this domain are not merely sites for the addition of palmitate groups, but play an essential role in membrane recognition before palmitoylation. Membrane association of the cysteine-string domain is not sufficient to trigger palmitoylation, which requires additional downstream residues that may regulate the membrane orientation of the cysteine-string domain. CSP palmitoylation-deficient mutants remain trapped in the ER, suggesting that palmitoylation may regulate ER exit and correct intracellular sorting of CSP. These results reveal a dual function of the cysteine-string domain: initial membrane binding and palmitoylation-dependent sorting.     

Smad signalling in the ovary

It has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGF-beta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.     

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

Summary Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25–35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.The assistance of A. Mazza and G.A.R. Mealing is gratefully acknowledged. The trypsin-activated, HPLC-purified CryIC toxin isolated from B. thuringiensis var. entomocidus crystal was a kind gift from M. Pusztai, Institute for Biological Sciences, NRC, Ottawa.     

Biology of the western pygmy perch,Edelia vittata,and comparisons with two other teleost species endemic to south-western Australia

Synopsis This paper provides data on the movements, spawning times and localities, size and age composition, growth and diet of the western pygmy perch,Edelia vittata, in a south-western Australian river. The results of this study are compared with those recorded previously for two other locally endemic species (Galaxias occidentalis andBostockia porosa) to ascertain the extent to which these three species utilise the same resources, and whether they are similarly adapted to the highly seasonal flow regime. A total of 3396Edelia vittata was collected with a 3 mm mesh seine net from the Collie River in south-western Australia at 3–6 week intervals over a period of 26 months. During the winter, fish moved from the river into either adjacent flood waters or tributary creeks. The trends shown by gonadosomatic indices, oocyte growth and prevalence of post-ovulatory follicles, demonstrate that spawning takes place between late winter and late spring (July–November). Sexual maturity was attained by both sexes at the end of the first year of life. Although 99% of the fish belonged to the 0+, 1+ and 2+ age classes, representatives of the 3+, 4+ and 5+ age classes were also caught. The respective von Bertalanffy growth curve parameters for L, K and t₀ were 54.9, 1.178 and -0.210 for males and 57.9, 1.124 and -0.198 for females. At the end of the first and second years of life, the lengths predicted from the von Bertalanffy growth equations were 42 mm ( 0.8 g) and 51 mm ( 1.4 g) for males and 43 mm ( 0.9 g) and 53 mm ( 1.6 g) for females. The diet consisted of a wide range of small benthic invertebrates, especially dipteran larvae, ostracods and cyclopoid copepods. The peak time, duration and predominant locality of spawning, the time of day when maximum activity occurs and the diet ofEdelia vittata differs from those previously recorded forGalaxias occidentalis andBostockia porosa. These differences would be likely to minimize any potential competition amongst these three species. The growth rates, age at first sexual maturity and pattern of oocyte development also differ amongst the three species.     

Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12, Salmonella typhimurium and Klebsiella pneumoniae. Physical association of enterochelin synthetase components in vitro

Further evidence is presented in support of the proposal made previously (Greenwood, K.T. and Luke, R.K.J. (1976) Biochim. Biophys. Acta 454, 285-297) that components of the Escherichia coli enterochelin synthetase system physicaloly associate to form enzyme complexes. Evidence for the existence of three enzyme complexes, designated in order of increasing stability G-D < F-D < F-D-G, has been obtained following gel filtration and chromatography on DEAE-Sephadex. Persistence of the F-D and G-D complexes during chromatography appears to depend on the flow rate of the column. On the basis of complementation with appropriate ent mutants of E. coli, activities corresponding to those of the D, E, F and G components of enterochelin synthetase in E. coli have been detected in cell-extracts of both Salmonella typhimurium and Klebsiella pneumoniae (formerly Aerobacter aerogenes) strains. These are designated D', E', F' and G' activities. Components E' and G' are eluted from Sephadex G-100 in similar fashion to their E. coli counterparts. Peaks of F' and D' activities however, are eluted together at a position corresponding to that of the E. coli F component. We suggest that in S. typhimurium and K. pneumoniae, either a single polypeptide combines the functions of the E. coli F and D components, or that separate F' and D' components form a stable complex and that activity of uncomplexed D' and component was not detected under the conditions used during chromatography and assay.     

Studies on the properties of a soluble phosphatidylinositol-phosphodiesterase of rabbit iris smooth muscle

Some properties of the soluble phosphatidylinositol phosphodiesterase (monophosphatidylinositol inositolphosphohydrolase, EC 3.1.4.10) of rabbit iris smooth muscle are described. Studies on its subcellular distribution showed that in this tissue the phosphodiesterase is not exclusively cytosolic. Thus, under our experimental conditions about 58% of the enzyme activity was found in the soluble fraction and the remainder was particulate. When the latter was treated with deoxycholate about 59% of the enzyme activity, compared to 86% of that of ATPase, was still bound to the particulate fraction. The kinetic properties of the enzyme (30--50% (NH4)2SO4 fraction) were examined. Maximum breakdown was 7.7 mumol/h per mg protein and occurred at pH 5.6. The products of [14C]arachidonic acid-labelled phosphatidylinositol were 1,2-diacylglycerol and a mixture of 86% myoinositol 1-phosphate and 14% myoinositol 1,2-(cyclic)phosphate. The enzyme has an absolute requirement for Ca2+. Addition of Ba2+, La3+, Mg2+, Mn2+, EGTA or EDTA at 0.05--5 mM concentrations; Sr2+ at higher concentrations (greater than 0.25 mM) markedly inhibited the phosphodiesterase activity and this inhibition was completely reversed by Ca2+. The enzyme is specific for the phosphoinositides.