Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P₂ fraction) from Fisher 344 female rats were incubated in the presence of ADP-chelated Fe³⁺ (0.5–50 M) and ascorbate (250 M). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by¹⁴CO₂ evolution froml-[1-¹⁴C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe³⁺/ascorbate was found with 50% inhibition occurring at 2.5 M Fe³⁺ concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe³⁺ in the absence of exogenous ascorbate was effective only above 25 M. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P₂ fractions using sucrose gradients. Membrane microviscosity of the purifled synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe³⁺/ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulhydryl content. It is concluded that ironinduced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.     

Acoustic observations of krill (Euphausia superba) at the ice edge (between elephant I. and south Orkney I., Dec. 1988/Jan. 1989)

The effect of the ice edge on Antarctic krill abundance, swarm parameters, distribution and migration, were investigated using acoustics. Two parameters, overall abundance and inter-swarm distance were found to increase with distance from the ice edge, while the number of swarms per unit distance decreased. Swarm dimensions, length and thickness do not seem to depend on proximity of ice. Krill near the ice-edge undergo diurnal vertical migration with a periodicity of 12 hours and an amplitude of about 6 m. Juvenile krill of 31 mm were dominant in the area investigated.     

Effects of Cortisol and Dexamethasone on Insulin Signalling Pathways in Skeletal Muscle of the Ovine Fetus during Late Gestation

Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth.     

Behavioural development of conspecific odour preferences in bank voles,Clethrionomys glareolus

Biological odours of conspecifics are known to have strong influences on behavioural interaction in bank voles Clethrionomys glareolus. This experiment tested two hypotheses. (1) Olfactory cues from familiar and unfamiliar mature opposite-sex conspecifics differ in their attractiveness to males and females, and their behavioural reactions change with age. (2) A genetically based mechanism is involved in female recognition of kin.In a two-choice preference test, prepubertal males and females were more attracted to familiar than to unfamiliar odours of opposite-sex conspecifics, as manifested by more time spent sniffing familiar voles. As the young reached sexual maturity they shifted their odour preferences. Mature males and females preferred the novel odour of unrelated opposite-sex conspecifics to that of relatives. The results of experiments testing the second hypothesis indicate that females use a genetically based mechanism to recognise their kin. Young and mature females were able to recognise the odour of their biological but socially unknown fathers, and showed the same pattern of behaviour as females in previous experiments.The possible biological functions of kin recognition in bank voles are discussed.     

Morphometric classification of hairy cell leukemia in bone marrow trephine biopsy

OBJECTIVE: To analyze cytologic and histologic parameters in bone marrow trephine biopsy in an attempt to define heterogeneity of hairy cell leukemia cells. STUDY DESIGN: The study group consisted of 28 trephine biopsies. Immunohistochemistry for CD20 antigen was used. Image processing and measurements were performed with AnalySIS 3.0 image analysis system (Soft Imaging System GmbH, Germany) and custom built programs. For planimetric measurements of nuclei, automatic segmentation was implemented. The measured parameters were: surface area, perimeter, minimum, mean and maximum diameter, and a set of form factors. Relative volumes of bone trabeculae, adipose tissue, hematopoietic tissue and neoplastic infiltrate were assessed by the point counting method. Nuclear volume was measured by the point sampled intercept method. Bone marrow fibrosis was assessed using a curvilinear line test system. RESULTS: Significant variability of cell nuclei was found, and their classification into 3 types was possible. The relative frequency of those types was different in various cases and allowed subdivision of cases into 3 groups that differed in some clinical and histologic manifestations. CONCLUSION: The present study demonstrated the heterogeneity of cell populations of hairy cell leukemia.     

Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.     

Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.