A1 toxicity in yeast. A role for Mg?

We have established conditions in which soluble Al is toxic to the yeast Saccharomyces cerevisiae. The major modifications to a standard synthetic medium were lowering the pH and the concentration of Mg ions. Alterations to the PO4, Ca, or K concentration had little effect on toxicity. Organic acids known to chelate Al reduced its toxicity, suggesting that Al3+ is the toxic Al species. The unique ability of Mg ions to ameliorate Al toxicity led us to investigate the hypothesis that Al inhibits Mg uptake by yeast. Yeast cells accumulate Mg, Co, Zn, Ni, and Mn ions via the same transport system (G.F. Fuhrmann, A. Rothstein [1968] Biochim Biophys Acta 163: 325-330). Al3+ inhibited the accumulation of 57Co2+ by yeast cells more effectively than Ga, La, or Mg. In addition, a mutant yeast strain with a defect in divalent cation uptake proved to be more sensitive to Al than a wild-type strain. Taken together, these results suggest that Al may cause Mg deficiency in yeast by blocking Mg transport. We discuss the relevance of yeast as a model for the study of Al toxicity in plant systems.     

A new angle on blood–CNS interfaces: A role for connexins?

Neuronal signaling in the CNS depends on the microenvironment around synapses and axons. To prevent fluctuations in blood composition affecting the interstitial fluid and CSF, two barriers, the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB), are interposed between the blood and the brain/CSF compartment. Brain capillary endothelial cells (ECs) constitute the BBB whereas choroid plexus epithelial (CPE) cells form the BCSFB. The anatomical basis of these barriers is located at the level of an intercellular junctional complex that impedes paracellular diffusion. Tight and adherens junctions are known as the principal constituents of this junctional complex. Transmembrane connexins (Cxs) are the prime building blocks of plasma membrane hemichannels that combine to form intercellular gap junctions (GJ). Although Cxs co-exist within the junctional complex, their influence on tight/adherens junctions and their role in barrier function of BBB ECs and CPE has been mostly ignored. Here, we review current knowledge on the role of Cxs in the BBB, BCSFB and other interfaces that subside within the CNS. We conclude that Cxs are a rather unexplored but promising target for influencing CNS barrier function.     

Vasodilatory effects of cholecystokinin: new role for an old peptide?

Cholecystokinin (CCK) peptides are involved in the control of multiple functions both in the central nervous system (CNS) and in the gastrointestinal tract where they act as neurotransmitters and regulate digestive functions. This review deals with the role of CCK peptides as vasoactive mediators. Recent work from our group demonstrates that CCK peptides induce neurogenic vasodilatation both in cerebral and mesenteric vessels. Such an effect is mediated by nitric oxide and seems to be presynaptic. These findings suggest that endogenous CCK peptides could be relevant vasodilatory agents involved in regulating both cerebral and splanchnic blood flow. We hypothesize here how such an effect could be useful in the interpretation of, in a new conceptual frame, the eventual contribution of CCK to some physiological and physiopathological events, such as splanchnic postprandial hyperaemia, panic attack or migraine.     

Molybdenum sequestration in Brassica species. A role for anthocyanins?

To elucidate plant mechanisms involved in molybdenum (Mo) sequestration and tolerance, Brassica spp. seedlings were supplied with molybdate, and the effects on plant physiology, morphology, and biochemistry were analyzed. When supplied with (colorless) molybdate Indian mustard (Brassica juncea) seedlings accumulated water-soluble blue crystals in their peripheral cell layers. Energy dispersive x-ray analysis showed that Mo accumulated predominantly in the vacuoles of the epidermal cells. Therefore, the blue crystals are likely to be a Mo compound. The x-ray absorption spectrum of the plant-accumulated Mo was different than that for molybdate, indicating complexation with a plant molecule. Because the blue compound was water soluble and showed a pH-dependent color change, possible involvement of anthocyanins was investigated. An anthocyanin-less mutant of Brassica rapa ("fast plants") was compared with varieties containing normal or high anthocyanin levels. The anthocyanin-less mutant did not show accumulation of a blue compound when supplied with molybdate. In the anthocyanin-containing varieties, the blue compound colocalized with anthocyanins in the peripheral cell layers. Mo accumulation by the three B. rapa varieties was positively correlated with anthocyanin content. Addition of molybdate to purified B. rapa anthocyanin resulted in an in vitro color change from pink to blue. Therefore, Mo appears to be sequestered in vacuoles of the peripheral cell layers of Brassica spp. as a blue compound, probably a Mo-anthocyanin complex.     

A role for skin in Ca metabolism of frogs?

• 1.1. In the leopard frog, Rana pipiens, in vivo Ca loss occurs in similar amounts across the skin and the urine. No change was detected in Ca loss when frogs were injected with either calcitonin or parathyroid hormone. Large doses of 1,25-(OH)₂vitamin D₃ increased urinary Ca loss.
• 2.2. ⁴⁵Ca accumulation across the skin in vivo each day is equivalent to 0.04% of total body Ca or 14% of the total Ca in the extracellular fluids. This accumulation was enhanced by prior adaptation of the frogs to a low Ca pond water.
• 3.3. Unidirectional influx (from 0.2 mM Ca on pond side) was remarkably similar in vitro: 0.56 and in vivo: 0.65 nmol cm⁻² h⁻¹. Based on in vitro measurements, an active transport process does not appear to be involved in this transcutaneous Ca movement.
• 4.4. Substantial deposits of Ca equivalent to five times the total in other “soft” tissues and eleven times that in the total extracellular fluids are found in R. pipiens skin.
• 5.5. Although cutaneous Ca does “turnover” slowly as shown by exchange with external ⁴⁵Ca. the skin Ca concentration does not change with the environmental Ca concentration.
• 6.6. Possible role(s) of cutaneous Ca in frogs' overall Ca metabolism are discussed.

     

Oxidative stress and potential free radical damage associated with photocopying. A role for ozone?

OBJECTIVE: To study the relationship between oxidative stress and potential free radical damage associated with photocopying and to explore a role for ozone emitted during the photocopying process. METHODS: 80 photocopying operators (PO) and 80 healthy volunteers (HV) were enrolled in a random control study design, in which the level of lipoperoxide (LPO, thiobarbituric acid reactive substances, TBARS) in erythrocytes and the levels of vitamin C (VC), vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as the activities of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes were determined by spectrophotometric methods. RESULTS: Compared with the average values of the above biochemical parameters in the HV group, the average value of LPO (TBARS) in erythrocytes in the PO group was significantly increased (P < 0.0001), while the average values of VC, VE and beta-CAR in plasma as well as those of SOD and CAT in erythrocytes in the PO group were significantly decreased (P < 0.0001). Pearson product-moment correlation analysis showed that with the increase of the ozone level in photocopying sites and the PO duration of exposure to ozone, the level of LPO in erythrocytes in the operators was increased (P < 0.001), while the levels of VC, VE and beta-CAR in plasma as well as the activities of SOD and CAT in erythrocytes in the operators were decreased (P < 0.01-0.0001). CONCLUSION: The findings in this study suggest that ozone causes oxidative damage in copier operatives.     

A therapeutic role for sirtuins in diseases of aging?

The sirtuins are a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Among the many genes that have been shown to affect aging in model organisms, sirtuin genes are unique in that their activity level is positively correlated with lifespan (i.e. they are anti-aging genes). Sirtuins are a druggable class of enzymes (i.e. amenable to intervention by small molecules) that could have beneficial effects on a variety of human diseases. In view of the many functions of Sirtuin 1 (SIRT1) in cells, this review focuses on its role in regulating important aspects of mitochondrial biology. Mitochondria have been linked to aging, and also to diseases of aging. Thus, sirtuins might provide a key link between mitochondrial dysfunction, aging and metabolic disease.     

Interaction of lipoprotein lipase with heparin–Sepharose. Evaluation of conditions for affinity binding

Lipoprotein lipases from a variety of sources have been shown previously to bind to heparin and some related polysaccharides. For the present studies lipoprotein lipase purified from bovine milk was used. 1. In batch experiments binding of the enzyme activity to heparin-Sepharose occurred relatively slowly, so that 30min was required for the system to come to near-equilibrium. In contrast, release of the enzyme activity from heparin-Sepharose by addition of salt to the liquid phase occurred rapidly. 2. Some binding was observed also with unsubstituted Sepharose, but this binding had a low capacity compared with that observed with heparin-Sepharose. High salt concentrations, heparin or deoxycholate decreased the binding to unsubstituted Sepharose. These factors also increase the solubility of the enzyme, which is low. 3. Addition of heparin to the liquid phase caused a concentration-dependent release of enzyme activity from the gel. These results suggested that the binding of the enzyme to heparin-Sepharose was mainly through interaction with heparin. 4. The enzyme activity was also quantitatively displaced to the liquid phase at increased concentrations of salt. Among the positive ions tested the following order of effectiveness was noted: Cs(+) approximately K(+)>Na(+)>Li(+); and among the negative the following: SCN(-)>I(-)> NO(3) (-)>Br(-) approximately Cl(-). The differences were quite large. Thus addition of 0.16m-KSCN (in addition to the 0.32m-NaCl originally present) displaced one-half of the enzyme activity to the supernatant, whereas 0.8m-LiCl only displaced one-quarter. 5. The distribution of heparin in the gel also profoundly influenced the binding. Two series of gels were studied. One series was made by mixing heparin-Sepharose with unsubstituted Sepharose. Results obtained with these gels were those expected from a series of decreasing volumes of heparin-Sepharose. In contrast, a series of heparin-Sepharoses made with different degrees of substitution gave quite different results. With these gels the amount of enzyme activity bound per amount of heparin increased markedly, whereas the salt concentration needed to displace the enzyme activity from the gel decreased markedly with decreased concentration of heparin in the gel. 6. On stepwise elution of small columns of heparin-Sepharose the enzyme activity was eluted over a remarkably wide range of salt concentrations. When enzyme eluted at one salt concentration was re-applied, it gave the same elution profile as enzyme previously eluted at other salt concentrations or the entire enzyme preparation. These and other results suggested that, whereas the enzyme preparation was rather homogeneous in its binding to heparin, the heparin preparation was polydisperse in binding of lipoprotein lipase.     

Detergent binding to unmyristylated protein kinase A—Structural implications for the role of Myristate

Myristylation often governs the targeting of protein kinases to the plasma membrane. It is now known that a key member of the src family of protein tyrosine kinases, pp60^v-src, binds to the lipid bilayer of the plasma membrane via a myristylated amino terminal sequence. The mechanism of this interaction is not known; however, myristic acid (Myristic acid may also be referred to as Myristate) and residues 2 through 14 are also absolutely required (Resh and Ling, 1990). This review presents an analysis of crystal structures of detergent-modified recombinant and myristylated mammalian catalytic subunit of protein kinase A. Crystals of unmyristylated recombinant catalytic subunit of protein kinase A are grown in the presence of Mega 8, a glucamide-type of detergent, and only this detergent binds, which results in a resolution extension (Knightonet al., 1991a). Comparisons of these two structures reveal that the detergent association with the recombinant enzyme binds in exactly the same hydrophobic pocket of the protein occupied by myristic acid in the mammalian protein (Karlssonet al., 1993; Zhenget al., 1993a). Removal of the detergent through soaking results in the local unwinding of the first helix, helix A, and disorder of the canonical recognition sequence of the phosphorylation site, Ser 10 (Zhenget al., 1993b). These results suggest that anchoring the myristic acid inside the protein results in formation of a stable structural template, which includes the myristylated amino terminal sequence important for the recognition by protein kinases. This inside out motif might provide a structural paradigm for the recognition of myristylated proteins, including pp60^v-src.     

A new predictor for the onset of atrial fibrillation?

In this issue of the Netherlands Heart Journal, the results of a substudy of the PREVEND trial are published.1 This prospective registry of presumably healthy citizens of Groningen aims to establish the association between microalbuminuria and the emergence of renal and cardiovascular diseases.2 This general population based long-term study extends the series of previous large-scale epidemiological trials conducted in the Netherlands. We recall among them the 1970 Vlagtwedde study for epidemiological cardiology and ischaemic heart disease, the 1982 Zutphen study of diet and cardiovascular diseases, the 1997 Maastricht study of circulatory arrest and sudden death, the 1998 Amsterdam study of out-of-hospital cardiac arrest and the 1999 Rotterdam study of prolonged QT interval and mortality.