Age-related changes in linoleate and alpha-linolenate desaturation by rat liver microsomes

The first and rate limiting step in the conversion of linoleic and alpha-linolenic acid is catalyzed by the delta - 6 - desaturase (D6D) enzyme. Rat liver microsomal D6D activity decreases on linolenic acid at a rate proportional to the animal age; on alpha-linolenic acid the decrease in D6D activity begins only later than on linoleic acid. The fatty acid composition of liver microsomes determined by gas chromatographic analysis confirms the impairment of the enzymatic activity directly measured. Our data indicate a correlation between aging and D6D activity impairment. The loss of D6D activity may be a key factor in aging through altering the eicosanoid balance.     

Aging influence on delta-6-desaturase activity and fatty acid composition of rat liver microsomes

The activity of delta-6-desaturase (D6D) in liver microsomes and fatty acid composition of microsomal lipids of rats of different ages were studied. The D6D activity was similar in suckling rats and in weaning rats. However, the enzyme showed a significantly decreased activity in oldest animals, and a linear correlation was found between the D6D activity and the animal age. The fatty acid composition data on total lipids of liver microsomes were consistent with the age-dependent changes in fatty acid desaturase activity. The major changes occurred in the linoleate and arachidonate fractions; the 20:4/18:2 ratio in liver microsomes decreased together with D6D activity during aging. The loss of D6D activity may be a key factor in aging through altering lipid membrane composition.     

Different effects of five dopamine receptor subtypes on nuclear factor-kappaB activity in NG108-15 cells and mouse brain

We previously showed that dopamine receptors D1R and D2R expressed in NG108-15 cells activated protein kinase A and extracellular signal-regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)-kappaB activity. To investigate whether other dopamine receptor subtypes regulate NF-kappaB, we established NG108-15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF-kappaB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF-kappaB-Luci activity, although forskolin-induced cyclic AMP responsive element-Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element-luciferase reporter not observed following stimulation with quinpirole in D3R- or D4R-expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF-kappaB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF-kappaB activity. By contrast, risperidone treatment enhanced NF-kappaB activity. Taken together, D2R and D1R/D5R had opposite effects on NF-kappaB activity in NG108-15 cells. Risperidone up-regulated and haloperidol down-regulated NF-kappaB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone.     

半胱胺对断奶前后仔猪胃粘膜H+-K+-ATPase表达和活性的影响

实验选取新生仔猪18窝, 随机分为实验组和对照组各9窝, 自12日龄起, 对照组饲喂基础乳猪料, 实验组在基础饲料中添加半胱胺120 mg/kg饲料, 仔猪均于35日龄断奶。分别于断奶前1 周、断奶当天、断奶后36 h、72 h、1周以及断奶后10 d屠宰仔猪取样, 每个时间点随机选取对照组和试验组各6头仔猪, 用相对定量RT PCR方法测定不同日龄仔猪胃组织中H K ATPase mRNA 表达的相对含量, 并同时测定H K ATPase的活性。结果表明: (1) 对照组仔猪在断奶前1 周至断奶后10 d, H K ATPase mRNA相对含量和H K ATPase活性有上升趋势, 两组仔猪在断奶后10 d H K ATPase mRNA相对含量和活性均达到最高水平, 但总体不表现显著的年龄差异; (2) 半胱胺能明显提高仔猪胃粘膜中H K ATPase mRNA的表达, 在断奶前1 周、断奶当天、断奶后1周和断奶后10 d均出现显著差异。半胱胺也能明显提高H K ATPase 的活性, 在断奶当天和断奶后10 d, H K ATPase 的活性分别被提高32 3%和18 3%; (3) 观察期内对照组和实验组H K ATPase mRNA水平和H K ATPase活性之间未发现显著的相关性。以上结果说明: 断奶前后仔猪H K ATPase的mRNA相对含量和活性没有明显的发育性变化, 断奶对H K ATPase mRNA表达和活性没有影响,而半胱     

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.     

D5 dopamine receptor regulation of reactive oxygen species production, NADPH oxidase, and blood pressure

Activation of D1-like receptors (D1 and/or D5) induces antioxidant responses; however, the mechanism(s) involved in their antioxidant actions are not known. We hypothesized that stimulation of the D5 receptor inhibits NADPH oxidase activity, and thus the production of reactive oxygen species (ROS). We investigated this issue in D5 receptor-deficient (D5-/-) and wild-type (D5+/+) mice. NADPH oxidase protein expression (gp91(phox), p47(phox), and Nox 4) and activity in kidney and brain, as well as plasma thiobarbituric acid-reactive substances (TBARS) were higher in D5-/- than in D5+/+ mice. Furthermore, apocynin, an NADPH oxidase inhibitor, normalized blood pressure, renal NADPH oxidase activity, and plasma TBARS in D5-/- mice. In HEK-293 cells that heterologously expressed human D5 receptor, its agonist fenoldopam decreased NADPH oxidase activity, expression of one of its subunits (gp91(phox)), and ROS production. The inhibitory effect of the D5 receptor activation on NADPH oxidase activity was independent of cAMP/PKA but was partially dependent on phospholipase D2. The ability of D5 receptor stimulation to decrease ROS production may explain, in part, the antihypertensive action of D5 receptor activation.     

Modulation of catalytic activity in multi-domain protein tyrosine phosphatases

Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs) containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1) domains, while the membrane-distal (D2) domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.     

Effect of starvation and insulin treatment on glycogen synthase D and synthase D phosphatase activity in rat heart

Summary We have previously shown that synthase phosphatase activity was decreased in starved animals and was rapidly restored by insulin administration (1). In order to determine whether the decreased phosphatase activity was due to a decrease in phosphatase enzyme per se or to a change in the substrate, synthase D, phosphatase activity has been determined using purified synthase D substrate. Using purified heart or liver synthase D, phosphatase activity was lower in extracts from starved animals than in fed animals. Insulin administration rapidly increased phosphatase activity in extracts from the starved animals. The total amount of endogenous synthase D which was convertible to synthase I was lower in extracts from starve animals, but this was rapidly increased within 15 minutes following insulin administration. These data suggest that starvation and insulin have a direct effect on the phosphatase enzyme activity per se and probably on the substrate suitability of synthase D as well.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

Identification of the catalytic residues of bifunctional glycogen debranching enzyme

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,4-->1,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain. To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis. Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed. The mutant enzymes showed the same purification profiles as that of wild-type enzyme on beta-CD-Sepharose-6B affinity chromatography. All the mutant enzymes possessed either transferase activity or glucosidase activity. Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity. In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

Site-directed mutagenesis of the calcium-binding site of α-amylase of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Amylases that are active under acidic conditions (pH <6), at higher temperatures (>70 degrees C) and have less reliance on Ca(2+) are required for starch hydrolysis. The alpha-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30 degrees C but a significantly improved specific activity at pH 5 and 70 degrees C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30 degrees C but had significantly less activity at 70 degrees C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30 degrees C but had an improved activity at 70 degrees C.     

Testosterone enhancement during pregnancy influences the 2D:4D ratio and open field motor activity of rat siblings in adulthood

In humans, the relationship between the prenatal testosterone exposure and the ratio of the second and the fourth digits (2D:4D) has been extensively studied. Surprisingly, data on this relationship have thus far been lacking in experimental animals such as rats. We studied the effect of maternal testosterone enhancement during pregnancy on the digit ratio and open field activity of adult progeny in Wistar rats. Elevated levels of maternal testosterone resulted in lower 2D:4D ratios and an elongated 4D on the left and right forepaws in both males and females. We found no sex difference in 2D:4D in control animals. In the open field test, control females were more active than control males and testosterone females, while the activity of testosterone females did not differ from that of control males. We found a positive correlation between motor activity and the right forepaw 2D:4D ratio of control males and females. Prenatal exposure to testosterone resulted in the disappearance of this correlation in both males and females. Our results show that elevated levels of testosterone during the prenatal period can influence forepaw 4D length, 2D:4D ratio, and open field motor activity of rats, and that these variables are positively correlated. Thus, this approach represents a noninvasive and robust method for evaluating the effects of prenatal testosterone enhancement on anatomical and physiological parameters.     

Structure-function analysis of the bacterial expansin EXLX1

We made use of EXLX1, an expansin from Bacillus subtilis, to investigate protein features essential for its plant cell wall binding and wall loosening activities. We found that the two expansin domains, D1 and D2, need to be linked for wall extension activity and that D2 mediates EXLX1 binding to whole cell walls and to cellulose via distinct residues on the D2 surface. Binding to cellulose is mediated by three aromatic residues arranged linearly on the putative binding surface that spans D1 and D2. Mutation of these three residues to alanine eliminated cellulose binding and concomitantly eliminated wall loosening activity measured either by cell wall extension or by weakening of filter paper but hardly affected binding to whole cell walls, which is mediated by basic residues located on other D2 surfaces. Mutation of these basic residues to glutamine reduced cell wall binding but not wall loosening activities. We propose domain D2 as the founding member of a new carbohydrate binding module family, CBM63, but its function in expansin activity apparently goes beyond simply anchoring D1 to the wall. Several polar residues on the putative binding surface of domain D1 are also important for activity, most notably Asp82, whose mutation to alanine or asparagine completely eliminated wall loosening activity. The functional insights based on this bacterial expansin may be extrapolated to the interactions of plant expansins with cell walls.     

Stable expression of immunoglobulin gene V(D)J recombinase activity by gene transfer into 3T3 fibroblasts

Using retroviral recombination substrates, we investigated the developmental stage of expression of V(D)J recombinase activity and the activation of recombinase activity in nonlymphoid cells. V(D)J recombinase activity was detected only in pre-B cells, but a DNA transfer protocol successfully activated V(D)J recombination in an NIH 3T3 cell line. The V(D)J recombinase activity was transferred in a second round of transfection and was then stably expressed in fibroblasts at a level comparable to that of a recombinationally active pre-B cell. It is likely that expression of a single, lymphoid-specific gene in a fibroblast is sufficient to confer V(D)J recombinase activity on that cell.     

Metalloproteinase activity in growth plate chondrocyte cultures is regulated by 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) and mediated through protein kinase C.

During endochondral development, growth plate chondrocytes must remodel their matrix in a number of ways as they differentiate and mature. In previous studies, we have shown that matrix metalloproteinases (MMPs) extracted from matrix vesicles can extensively degrade aggrecan and that this is modulated by vitamin D metabolites in a manner involving protein kinase C (PKC). Matrix vesicles represent only a small component of the extracellular matrix, however, and it is unknown if the total metalloproteinase complement, including the MMPs and aggrecanases in the culture, is also regulated in a similar way. This study tested the hypothesis that vitamin D metabolites regulate the level of metalloproteinase activity in growth plate chondrocytes via a PKC-dependent mechanism and play a role in partitioning this proteinase activity between the media and cell layer (cells+matrix) in these cultures. To do this, resting zone cells (RC) were treated with 10(-9)-10(-7) M 24R,25-(OH)(2)D(3), while growth zone cells (GC) were treated with 10(-10)-10(-8) M 1alpha,25-(OH)(2)D(3). Cultures of both cell types were also treated with the PKC inhibitor chelerythrine in the presence and absence of vitamin D metabolites. At harvest, the media were either left untreated or treated to destroy metalloproteinase inhibitors, while enzyme activity in the cell layers was extracted with buffered guanidine and then treated like the media to destroy metalloproteinase inhibitors. Neutral metalloproteinase (aggrecan-degrading activity) activity was assayed on aggrecan-containing polyacrylamide gel beads and collagenase activity was measured on telopeptide-free type I collagen. Neutral metalloproteinase activity was found primarily in the cell layer of both cell types; however, activity was greater in extracts of GC cell layers. No collagenase activity could be detected in RC extracts until the metalloproteinase inhibitors were destroyed. In contrast, extracts of GC cell layers contained measurable activity without removing the inhibitors, and destroying the inhibitors resulted in a greater than two-fold increase in activity. No collagenase activity was found in the media of either cell type. 24,25-(OH)(2)D(3) caused a dose-dependent increase in neutral metalloproteinase activity in extracts of RC cells, but had no effect on collagenase activity. In contrast, 1,25-(OH)(2)D(3) caused a dose-dependent decrease in collagenase activity in extracts of GC cells, but had no effect on neutral metalloproteinase activity. In both cases, the effect of the vitamin D metabolite was mediated through the activation of PKC. These results support the hypothesis that metalloproteinases are involved in regulating the bulk turnover of collagen and aggrecan in growth plate chondrocytes and that the amount of metalloproteinase activity found is a function of the cell maturation state. Furthermore, 83-93% of neutral metalloproteinase activity and 100% of collagenase activity is localized to the cell layer. Moreover, the regulation of metalloproteinase activity by 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) involves a PKC-dependent pathway that is controlled by the target cell-specific vitamin D metabolite.     

Intestinal Na(+)/Ca(2+) exchanger protein and gene expression are regulated by 1,25(OH)(2)D(3) in vitamin D-deficient chicks

The role of 1,25(OH)(2)D(3) on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)(2)D(3) were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)(2)D(3) simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)(2)D(3). NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)(2)D(3). Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)(2)D(3) treatment. Rapid effects of 1,25(OH)(2)D(3) on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)(2)D(3) on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)(2)D(3) enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

Studies on the leukotriene D4-metabolizing enzyme of rat leukocytes, which catalyzes the conversion of leukotriene D4 to leukotriene E4

Leukotriene D4-metabolizing enzyme was studied using rat neutrophils, lymphocytes and macrophages. These leukocyte sonicates converted leukotriene D4 to leukotriene E4. However, the leukotriene D4-metabolizing activity varied with cell type, and macrophages showed the highest activity among these leukocytes. The subcellular localization of the leukotriene D4-metabolizing enzyme of macrophages was examined, and the leukotriene D4-metabolizing activity was found to be present in the membrane fraction, but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the leukotriene D4-metabolizing activity of macrophages decreased significantly (about 95%) without any inhibition of marker enzymes of microsome, cytosol, lysosome and mitochondria. When neutrophils and lymphocytes were modified with diazotized sulfanilic acid, the leukotriene D4-metabolizing activity was also inhibited about 90% by the modification. Among various enzyme inhibitors used, o-phenanthroline, a metal chelator, remarkably inhibited the leukotriene D4-metabolizing activity of leukocytes, and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+ and Zn2+. These findings seem to indicate that rat neutrophils, lymphocytes and macrophages possess the leukotriene D4-metabolizing metalloenzyme which converts leukotriene D4 to leukotriene E4, on the cell surface, although macrophages have a higher enzyme activity than the other two.     

Spring Time and Fall Time Effects of 1,25-Dihydroxyvitamin D3, Cobalt (II) and (CoCl2(1,25(OH)2D3)4) Complex on Renal and Cerebral Enzymatic Markers in Rats

In this work, we studied the chronesthesia of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), cobalt (II) chloride and of the complex [CoCl 2 (1,25(OH) 2 D 3 ) 4 ]. The study was carried out in spring and autumn on AP and ?-GT activities in brain and kidney of rats during the rodents' active period. In rat brain, in both seasons, 1,25(OH) 2 D 3 enhanced AP activity by 59 % in spring and 21 % in autumn and ?-GT by 39 and 35 % respectively. Cobalt (II) stimulated AP activity by 34 and 29 % respectively. The complex was mainly active on ?-GT activity (70 and 36 %) showing a synergic effect on ?-GT activity in June. In rat kidney, during spring, the induction of AP activity for 1,25(OH) 2 D 3 , cobalt (II) chloride and their complex was, respectively 21, 18 and 12 %. The ?-GT activity was not modified during this period, whereas in autumn, it was inhibited by -33, -50 and -28 %. The AP activity in autumn was not altered. We conclude that the effects on the two enzymatic markers of the three compounds 1,25(OH) 2 D 3 , cobalt (II) chloride and [CoCl 2 (1,25(OH) 2 D 3 ) 4 ] are quite different in Spring and Autumn, and this is explained on the basis of chronesthesia.