Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.     

Identification and characterization of beta1-adrenergic receptors in rat parotid membranes

• Beta-adrenergic receptor identification and properties are probed in rat parotid membranes utilizing the high affinity β-adrenergic antagonist(−)-[³H]dihydroalprenolol.

• The binding of (−)-[³H]dihydroalprenolol to membrane preparations of parotid is rapid, equilibrium being reached in 5 min. Strict stereospecificity is observed, (−)-propanolol being 100 times more potent than (+)-propranolol in competing with (−)-[³H]dihydroalprenolol for binding sites. Beta-adrenergic agonists compete for the binding sites with (−)-[³H]dihydroalprenolol with the same characteristics, i.e., much higher concentrations of the (+)-stereoisomers than the (−)-stereoisomers are required to produce 50% inhibition, the range varies from 14-fold for epinephrine to 300-fold for isoproterenol. Among the (−)-stereoisomers, the relative potency of inhibitory action is (−)-propranolol > (−)-isoproterenol > (−)-epinephrine ≡ (−)-norepinephrine. (−)-Isoproterenol is about 20 times as potent as norepinephrine, the least potent agonist among all the catecholamine (−)-stereoisomers.

• The binding of (−)-[³H]dihydroalprenolol is saturable, with a maximum number of binding sites equalling 450 fmol/mg protein and a dissociation constant of 7.9 nM. The Scatchard plots show no significant curvilinear character. Hill plots consistently give a Hill coefficient close to unity (0.92–1.05). Both pieces of evidence suggest a single-component system with no significant cooperativity.

• Dissociation kinetics study after the method of De Metys et al. (1973) Biochem. Biophys. Res. Commun. 155, 154, indicates a lack of site-to-site interactions among the binding sites. The rate of dissociation of bound (−)-[³H]dihydroalprenolol is the same in the presence and absence of 1 · 10⁻⁵ M (±)-alprenolol.

• Based on the binding of (−)-[³H]dihydroalprenolol, it is concluded that the beta-adrenergic receptors can be identified in rat parotid and that these binding sites display β₁ character. Results of the study indicate a one-component system with no observable site-to-site interactions.

Lipid and fatty acid yield of nine stationary-phase microalgae: Applications and unusual C<Subscript>24</Subscript>–C<Subscript>28</Subscript> polyunsaturated fatty acids

Nine microalgal species from the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae and Dinophyceae were isolated from Australian waters, cultured to stationary phase and analyzed for their lipid and fatty acid composition and yield. Five species (Pavlova pinguis, Heterocapsa niei, Proteomonas sulcata, Navicula jeffreyi and Thalassiosira pseudonana) produced high proportions of triacylglycerol (TAG: 22–57% total lipid). An unidentified Navicula-like diatom (CS-786), despite having a low TAG content, had the highest EPA yield (5.8 mg L⁻¹), due to high biomass and a high relative proportion of EPA. Heterocapsa niei had the highest DHA yield (2.9 mg L⁻¹), due to a high cellular lipid and DHA content (171 pg cell⁻¹ and 13.7 pg cell⁻¹, respectively) despite its relatively low biomass. The desirable PUFA composition and yield of both diatom CS-786 and H. niei make them potential candidates for optimization of biomass and PUFA production for use as live-feeds in aquaculture. In addition, H. niei may have potential as a source of DHA for other uses. Low proportions (< 1.2%) of 24:6(n−3) accompanied by trace proportions of 24:5(n−6) were detected in most strains, while 28:8(n−3) was found in dinoflagellates and also in the prymnesiophyte P. pinguis. All non-diatomaceous species contained 26:7(n−3) in minor quantities. This is the first time these unusual C₂₄ and C₂₆ PUFA have been reported in microalgae and the first report of C₂₈ PUFA in a microalga other than dinoflagellates. Possible biosynthetic reasons why these might occur in stationary phase cultures are considered and the likely dietary transfer of these PUFA to higher aquatic life is discussed.     

Understanding the local actions of lipids in bone physiology

The adult skeleton is a metabolically active organ system that undergoes continuous remodeling to remove old and/or stressed bone (resorption) and replace it with new bone (formation) in order to maintain a constant bone mass and preserve bone strength from micro-damage accumulation. In that remodeling process, cellular balances – adipocytogenesis/osteoblastogenesis and osteoblastogenesis/osteoclastogenesis – are critical and tightly controlled by many factors, including lipids as discussed in the present review.Interest in the bone lipid area has increased as a result of in vivo evidences indicating a reciprocal relationship between bone mass and marrow adiposity. Lipids in bones are usually assumed to be present only in the bone marrow. However, the mineralized bone tissue itself also contains small amounts of lipids which might play an important role in bone physiology. Fatty acids, cholesterol, phospholipids and several endogenous metabolites (i.e., prostaglandins, oxysterols) have been purported to act on bone cell survival and functions, the bone mineralization process, and critical signaling pathways. Thus, they can be regarded as regulatory molecules important in bone health. Recently, several specific lipids derived from membrane phospholipids (i.e., sphingosine-1-phosphate, lysophosphatidic acid and different fatty acid amides) have emerged as important mediators in bone physiology and the number of such molecules will probably increase in the near future. The present paper reviews the current knowledge about: (1°) bone lipid composition in both bone marrow and mineralized tissue compartments, and (2°) local actions of lipids on bone physiology in relation to their metabolism. Understanding the roles of lipids in bone is essential to knowing how an imbalance in their signaling pathways might contribute to bone pathologies, such as osteoporosis.     

Role of labile iron in the toxicity of pharmacological ascorbate

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H₂O₂ via the formation of ascorbate radical (Asc^•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H₂O₂ generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H₂O₂ generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe³⁺/Fe²⁺, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.     

Effects of birth weight and maternal dietary fat source on the fatty acid profile of piglet tissue

This study aimed to investigate the effects and possible interactions of birth weight and n-3 polyunsaturated fatty acid (PUFA) supplementation of the maternal diet on the fatty acid status of different tissues of newborn piglets. These effects are of interest as both parameters have been associated with pre-weaning mortality. Sows were fed a palm oil diet or a diet containing 1% linseed, echium or fish oil from day 73 of gestation. As fish oil becomes a scarce resource, linseed and echium oil were supplemented as sustainable alternatives, adding precursor fatty acids for DHA to the diet. At birth, the lightest and heaviest male piglet per litter were killed and samples from liver, brain and muscle were taken for fatty acid analysis. Piglets that died pre-weaning had lower birth weights than piglets surviving lactation (1.27±0.04 v. 1.55±0.02 kg; P<0.001), but no effect of diet on mortality was found. Lower DHA concentrations were observed in the brain of the lighter piglets compared with their heavier littermates (9.46±0.05 v. 9.63±0.04 g DHA/100 g fatty acids; P=0.008), suggesting that the higher incidence of pre-weaning mortality in low birth weight piglets may be related to their lower brain DHA status. Adding n-3 PUFA to the sow diet could not significantly reduce this difference in DHA status, although numerically the difference in the brain DHA concentration between the piglet weight groups was smaller when fish oil was included in the sow diet. Independent of birth weight, echium or linseed oil in the sow diet increased the DHA concentration of the piglet tissues to the same extent, but the concentrations were not as high as when fish oil was fed.     

Feeding habitat and silvering stage affect lipid content and fatty acid composition of European eel Anguilla anguilla tissues

Lipids, particularly fatty acids (FAs), are major sources of energy and nutrients in aquatic ecosystems and play key roles during vertebrate development. The European eel Anguilla anguilla goes through major biochemical and physiological changes throughout its lifecycle as it inhabits sea- (SW), and/or brackish- (BW) and/or freshwater (FW) habitats. With the ultimate goal being to understand the reasons for eels adopting a certain life history strategy (FW or SW residency vs. ‘habitat shifting’), we explored differences in lipid content and FA composition of muscle, liver and eyes from eels collected across Norwegian SW, BW and FW habitats, and at different lifecycle stages (yellow to silver). FW and SW eels had a higher lipid content overall compared to BW eels, reflecting differences in food availability and life history strategies. SW eels had higher proportions of certain monounsaturated FAs (MUFAs; 18:1n-9, 20:1n-9), and of the essential polyunsaturated FAs 20:5n-3 (eicosapentaenoic acid, EPA) and 22:6n-3 (docosahexaenoic acid) than FW eels, reflecting a marine-based diet. In contrast, the muscle of FW eels had higher proportions of 18:3n-3, 18:2n-6 and 20:4n-6 (arachidonic acid), as is typical of FW organisms. MUFA proportions increased in later stage eels, consistent with the hypothesis that the eels accumulate energy stores prior to migration. In addition, the decrease of EPA with advancing stage may be associated with the critical role that this FA plays in eel sexual development. Lipid and FA information provided further understanding of the habitat use and overall ecology of this critically endangered species.     

Induction of apoptosis and lipogenesis in human preadipocyte cell line by n −3 PUFAs

We examined the effect of n ?3 PUFAs (polyunsaturated fatty acids) on the growth and maturation of human preadipocyte cell line AML‐I. On day 3 of the culture, n ?3 fatty acids such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), but not n ?6 fatty acid LA (linoleic acid), induced growth arrest accompanied by the appearance of characteristics of apoptosis in AML‐I cells at concentrations between 250 and 500 μM by Annexin V‐FITC staining. In Western blotting analysis, the loss of NF‐κB, Bcl‐2 and p‐Akt and the accumulation of Bad and Akt were observed in the cytoplasmic protein from the EPA‐treated cells. Exposure of AML‐I to EPA or DHA increased the cytoplasmic lipid accumulation compared with the vehicle‐treated cells in a time‐dependent manner during 4 and 6 days culture period by Oil Red O staining. The expression of FAS (fatty acid synthase) and PPAR‐γ (peroxisome proliferator‐activated receptor‐γ) were increased in EPA‐treated cells. These results suggest that EPA and DHA promote differentiation, inhibit proliferation and induce apoptosis in preadipocyte cell line AML‐I.     

HPLC示差折光法测定藻油中DHA的含量

首次采用HPLC示差折光法测定藻油中DHA的含量。色谱柱为Eclipse XDB-C18(Analytical 4.6×250mm,5μm),流动相为乙腈∶四氢呋喃∶水(含0.4%HAC)=77∶3∶20,柱温为30℃,流速为1.0 mL/min。该条件下DHA在0.259～6.225 mg/mL内呈良好的线性关系,相关系数R2=0.9995,平均回收率为101.15%,RSD为1.98%(n=6)。该方法可靠、简便、重现性好,可作为藻油中DHA的定量方法。