Identification of a protein component of the Ca2+-dependent K+ channel by affinity labelling with apamin

The red blood cell membrane proteins and plasma proteins of normal and spontaneously hypertensive rats were studied by uni- and bidimensional polyacrylamide gel electrophoresis. The amount of band 3, the major intrinsic protein of the erythrocyte membrane, was observed to be significantly reduced in spontaneously hypertensive rats. Plasma from these rats contained two additional heat stable proteins, characterized by a molecular weight of 16,000 daltons and isoelectric points of 4.7 and 5.1, respectively. These proteins were not detected in normotensive control Wistar Kyoto rats, in normal Wistar rats, or in Wistar rats with experimentally induced hypertension.     

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.     

Immune response of macrophages from young and aged mice to the oral pathogenic bacterium <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1β, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg.     

A plasminogen-like protein selectively degrades stearoyl-CoA desaturase in liver microsomes

Stearoyl-CoA desaturase (SCD) is an integral membrane protein of the endoplasmic reticulum that is rapidly and selectively degraded when isolated liver microsomes are incubated at 37 degrees C. We previously reported the purification of a 90-kDa microsomal protein with SCD protease activity and characterized the inhibitor sensitivity of the protease. Here we show that the 90-kDa protein is a microsomal form of plasminogen (Pg) and that the purified SCD protease contains a spectrum of plasmin-like derivatives. The 90-kDa protein was identified as Pg by mass spectrometry of its tryptic peptides. The purified SCD protease reacted with Pg antibody, and immunoblotting demonstrated enrichment of Pg by the purification procedure established for the SCD protease. Analysis of microsomes by zymography demonstrated a single band of proteolytic activity at 70-kDa corresponding to the mobility of Pg in nonreduced polyacrylamide gels. When microsomes were incubated at 37 degrees C prior to zymography, an intense band of proteolytic activity developed at 30-kDa. The purified SCD protease displayed a spectrum of proteolytic bands ranging from 70 to 30 kDa. Degradation of SCD by the purified protease and by microsomes was inhibited by bdellin, a plasmin inhibitor from the medicinal leech Hirudo medicinalis. To explore the role of Pg in the degradation of SCD in vivo, we examined SCD expression and degradation in microsomes isolated from Pg-deficient (Pg-/-) mice. Compared with microsomes from wild-type littermate control mice, liver microsomes from Pg-/- mice had significantly higher levels of SCD. Degradation of SCD in microsomes from Pg-/- mice was markedly diminished, whereas liver microsomes from control mice showed rapid SCD degradation similar to that observed in rat liver microsomes. These findings indicate that SCD is degraded by a protease related to Pg and suggest that plasmin moonlights as an intracellular protease.     

Identification of the stem rust resistance gene Pg9 and its association with crown rust resistance and endosperm proteins in 'Dumont' oat.

The Canadian oat cultivar 'Dumont' is known to have genes Pc38 and Pc39 for crown rust resistance and genes Pg2 and Pg13 for stem rust resistance. When crossed to a susceptible oat line OT328, 'Dumont' was shown to have an additional dominant gene for crown rust resistance, designated PcX. Tests of segregating progeny indicated that the stem rust resistance gene Pg9 is present and is tightly linked in coupling to PcX. The presence of Pg9 in 'Dumont' was confirmed in crosses involving the cultivar 'Ukraine', which has Pg9 and a crown rust resistance gene tightly linked to it. The association of rust resistance with endosperm proteins in 'Dumont' was investigated. The linkage of gene Pg13 with a 56.6-kDa polypeptide locus (map distance of 10.47 +/- 2.70 cM) was demonstrated using sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE). A 27.9-kDa polypeptide was shown to be associated with the linked PcX/Pg9 loci by SDS-PAGE but appeared to be more reliably separated as an avenin band, designated B4, using acid-PAGE. Another avenin band, designated B2, also was shown to be associated with the PcX/Pg9 loci using acid-PAGE. The loci conditioning the B2 and B4 bands appeared to be tightly linked or allelic and are separated from the linked PcX/Pg9 loci by a map distance of 1.03 +/- 0.36 cM. The association of Pg13 with a 56.6-kDa polypeptide and the tight linkage between PcX/Pg9 and the B2 (in coupling) and B4 (in repulsion) avenin loci offer a useful tool to breeders to detect the presence of these genes in oat breeding.     

Myocardial hypertrophy of normotensive Wistar-Kyoto rats

In our studies with spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Wistar rats, we observed normotensive WKY rats with cardiac hypertrophy determined by a greater left ventricular (LV) mass (LVM)-to-body weight (BW) ratio (LVM/BW) than that of normotensive Wistar rats. Thus we compared the following parameters in SHR, WKY, and Wistar rats: LVM/BW, cell capacitance as index of total surface area of the myocytes, length, width, and cross-sectional area of cardiac myocytes, LV collagen volume fraction, and myocardial stiffness. The LVM/BW of WKY (2.41 +/- 0.03 mg/g, n = 41) was intermediate between SHR (2.82 +/- 0.04 mg/g, n = 47) and Wistar rats (1.98 +/- 0.04 mg/g, n = 28). A positive correlation between blood pressure and LVM was found in SHR, whereas no such relationship was observed in WKY or Wistar rats. Cell capacitance and cross-sectional area were not significantly different in SHR and WKY rats; these values were significantly higher than those of Wistar rats. The cell length was smaller but the width was similar in WKY compared with SHR. Papillary muscles isolated from the LV of WKY and SHR were stiffer than those from Wistar rats. Consistently, a greater level of myocardial fibrosis was detected in WKY and SHR compared with Wistar rats. These findings demonstrate blood pressure-independent cardiac hypertrophy in normotensive WKY rats.     

Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions

Binding of streptokinase (SK) to plasminogen (Pg) activates the zymogen conformationally and initiates its conversion into the fibrinolytic proteinase, plasmin (Pm). Equilibrium binding studies of SK interactions with a homologous series of catalytic site-labeled fluorescent Pg and Pm analogues were performed to resolve the contributions of lysine binding site interactions, associated changes between extended and compact conformations of Pg, and activation of the proteinase domain to the affinity for SK. SK bound to fluorescein-labeled [Glu]Pg(1) and [Lys]Pg(1) with dissociation constants of 624 +/- 112 and 38 +/- 5 nM, respectively, whereas labeled [Lys]Pm(1) bound with a 57000-fold tighter dissociation constant of 11 +/- 2 pM. Saturation of lysine binding sites with 6-aminohexanoic acid had no effect on SK binding to labeled [Glu]Pg(1), but weakened binding to labeled [Lys]Pg(1) and [Lys]Pm(1) 31- and 20-fold, respectively. At low Cl(-) concentrations, where [Glu]Pg assumes the extended conformation without occupation of lysine binding sites, a 23-fold increase in the affinity of SK for labeled [Glu]Pg(1) was observed, which was quantitatively accounted for by expression of new lysine binding site interactions. The results support the conclusion that the SK affinity for the fluorescent Pg and Pm analogues is enhanced 13-16-fold by conversion of labeled [Glu]Pg to the extended conformation of the [Lys]Pg derivative as a result of lysine binding site interactions, and is enhanced 3100-3500-fold further by the increased affinity of SK for the activated proteinase domain. The results imply that binding of SK to [Glu]Pg results in transition of [Glu]Pg to an extended conformation in an early event in the SK activation mechanism.     

Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilizes the active zymogen conformation

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.     

24-Hour Changes in Circulating Prolactin,Follicle-Stimulating Hormone,Luteinizing Hormone,and Testosterone in Young Male Rats Subjected to Calorie Restriction

This work analyzes the effect of calorie restriction on the 24 h variation of pituitary-testicular function in young male Wistar rats by measuring the circulating levels of prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Control animals were provided an equilibrium calorie diet and the experimental animals a calorie-restriction diet equivalent to 66% of food restriction for four weeks starting on day 35 of life. Different groups of control and experimental rats were killed at 6 h intervals around the clock, beginning 1 h after light on (HALO). Compared to the control animals, the mean secretion of prolactin was augmented and that of LH and testosterone decreased in calorie-restricted rats, whereas FSH release remained unchanged. Significant changes in the 24 h secretory pattern of circulating prolactin, LH, and testosterone occurred in the calorie-restricted rats. These include the appearance of a second maximum of plasma prolactin at 21 HALO, blunting of the LH peak seen at 13 HALO, and phase-shift of the testosterone peak from 13 HALO in controls to 17 HALO in calorie-restricted rats. The significant positive correlation between individual LH and testosterone levels found in controls was no longer observed in calorie-restricted rats. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of the pituitary-gonadal axis in growing male rats.     

Enhancement through mutagenesis of the binding of the isolated kringle 2 domain of human plasminogen to omega-amino acid ligands and to an internal sequence of a Streptococcal surface protein.

In the background of the recombinant K2 module of human plasminogen (K2(Pg)), a triple mutant, K2(Pg)[C4G/E56D/L72Y], was generated and expressed in Pichia pastoris cells in yields exceeding 100 mg/liter. The binding affinities of a series of lysine analogs, viz. 4-aminobutyric acid, 5-aminopentanoic acid, epsilon-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyclohexane-1-carboxylic acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2(Pg) (K2(Pg)[C4G]). The variant, K2(Pg)[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2(Pg)[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2(Pg)[C4G]. The thermal stability of K2(Pg)[C4G/E56D/L72Y] was increased by approximately 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence of up-regulating the lysine binding property of K2(Pg) was explored, as reflected by its ability to interact with an internal sequence of a plasminogen-binding protein (PAM) on the surface of group A streptococci. A 30-mer peptide of PAM, containing its K2(Pg)-specific binding region, was synthesized, and its binding to each mutant of K2(Pg) was assessed. Only a slight enhancement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K(d) = 460 nM). A 5-fold decrease in binding affinity was observed for K2(Pg)[C4G/L72Y] (K(d) = 2200 nM). However, a 12-fold enhancement in binding to this peptide was observed for K2(Pg)[C4G/E56D/L72Y] (K(d) = 37 nM). Results of these PAM peptide binding studies parallel results of omega-amino acid binding to these K2(Pg) mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2(Pg), occurs through its lysine-binding site. This conclusion is supported by the 100-fold decrease in PAM peptide binding to K2(Pg)[C4G/E56D/L72Y] in the presence of 50 mM 6-aminohexanoic acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp(56) and Tyr(72) in the K2(Pg)[C4G/E56D/L72Y] mutant are synergistically coupled in terms of their contribution to the enhancement of PAM peptide binding.     

近交系HFJ大鼠的抗肿瘤细胞Walker-256特性

目的检测近交系HFJ大鼠的肿瘤学特性。方法采用大鼠肿瘤细胞Walker-256分别接种HFJ大鼠和Wistar大鼠制作腹水瘤、实体瘤模型,观察两种动物对同一种肿瘤细胞的敏感性及免疫反应差异。结果对于腹水瘤Walker-256接种7d,Wistar大鼠有6只腹水产生为阴性,HFJ大鼠腹水产生均为阳性。继续观察至20d,可见到Wistar大鼠有3只腹水阴性(阳性率9/12,死亡2只,染色体用1只),而HFJ大鼠腹水全部为阴性(阳性率0/12,死亡2只,染色体用1只)。腹水中Walker-256细胞染色体分析,Wistar大鼠和HFJ大鼠众数变化范围均为50～62条,无显著差异。实体瘤接种7d,Wistar大鼠和HFJ大鼠均可触摸到右侧腋下有肿块产生。20d后Wistar大鼠除2只肿块消失外其他均有肿块存在并随时间延长而增大(阳性率13/15);所有HFJ大鼠腋下肿块均变软并逐渐消失(阳性率0/15)。检测各组大鼠的细胞免疫、体液免疫功能,发现正常HFJ大鼠IgM、IgA显著低于Wistar大鼠(P<0.01),IgG差异不显著。荷瘤组HFJ大鼠和Wistar大鼠IgG均高于各自正常对照组,差异极显著(P<0.01)。Wist-ar大鼠腹水瘤阳性组IgG显著低于阴性组和HFJ腹水阳性组(P<0.05),Wistar大鼠实体瘤阳性组也显著低于HFJ实体阳性组(P<0.01)。Wistar大鼠腹水瘤阳性组IgM显著低于阴性组(P<0.05),Wistar大鼠腹水瘤阴性组和HFJ大鼠腹水瘤、实体瘤阳性组IgM均高于各自对照组,且差异显著(P<0.05)。细胞免疫结果显示各组CD4+数量差异不显著;正常HFJ大鼠CD8+显著少于Wistar大鼠(P<0.05),Wistar大鼠腹水瘤和实体瘤阳性组CD8+数量较阴性组和正常对照组均显著减少(P<0.05)。各荷瘤阴性组大鼠CD8+数量均较正常值增加,除Wistar大鼠腹水瘤和HFJ实体瘤阴性组大鼠差异显著(P<0.05)外,其他均不显著;CD4+/CD8+结果与CD8+相反。结论 HFJ大鼠具有抗大鼠肿瘤细胞Walker-256的特性,腹水瘤及实体瘤均不易生长。     

Plasminogen carbohydrate side chains in receptor binding and enzyme activation: a study of C6 glioma cells and primary cultures of rat hepatocytes.

The human [Glu1]-plasminogen carbohydrate isozymes, plasminogen type I (Pg 1) and plasminogen type II (Pg 2), were separated by chromatography and studied in cell binding experiments at 4 degrees C with primary cultures of rat hepatocytes and rat C6 glioma cells. In both cell systems, Pg 1 and Pg 2 bound to an equivalent number of receptors, apparently representing the same population of surface molecules. The affinity for Pg 2 was slightly higher. With hepatocytes, the KD for Pg 1 was 3.2 +/- 0.2 microM, and the KD for Pg 2 was 1.9 +/- 0.1 microM, as determined from Scatchard transformations of the binding isotherms. The Bmax was approximately the same for both isozymes. With C6 cells, the KD for Pg 1 was 2.2 +/- 0.1 microM vs. 1.5 +/- 0.2 microM for Pg 2. Again, the Bmax was similar with both isozymes. 125I-Pg 1 and 125I-Pg 2 were displaced from specific binding sites by either nonradiolabeled isozyme. The KI for Pg 2 was slightly lower than the KI for Pg 1 with hepatocytes (0.9 vs. 1.3 microM) and with C6 cells (0.6 vs. 1.1 microM). No displacement was detected with miniplasminogen at concentrations up to 5.0 microM. Activation of Pg 1 and Pg 2 by recombinant two-chain tissue-plasminogen activator (rt-PA) was enhanced by hepatocyte cultures. The enhancing effect was greater with Pg 2. Hepatocyte cultures did not affect the activation of miniplasminogen by rt-PA or the activation of plasminogen by streptokinase. Unlike the hepatocytes, C6 cells did not enhance the activation of plasminogen by rt-PA or streptokinase; however, plasmin generated in the presence of C6 cells reacted less readily with alpha 2-antiplasmin.     

Duodenal-jejunal bypass protects GK rats from {beta}-cell loss and aggravation of hyperglycemia and increases enteroendocrine cells coexpressing GIP and GLP-1

Dramatic improvement of type 2 diabetes is commonly observed after bariatric surgery. However, the mechanisms behind the alterations in glucose homeostasis are still elusive. We examined the effect of duodenal-jejunal bypass (DJB), which maintains the gastric volume intact while bypassing the entire duodenum and the proximal jejunum, on glycemic control, β-cell mass, islet morphology, and changes in enteroendocrine cell populations in nonobese diabetic Goto-Kakizaki (GK) rats and nondiabetic control Wistar rats. We performed DJB or sham surgery in GK and Wistar rats. Blood glucose levels and glucose tolerance were monitored, and the plasma insulin, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) levels were measured. β-Cell area, islet fibrosis, intestinal morphology, and the density of enteroendocrine cells expressing GLP-1 and/or GIP were quantified. Improved postprandial glycemia was observed from 3 mo after DJB in diabetic GK rats, persisting until 12 mo after surgery. Compared with the sham-GK rats, the DJB-GK rats had an increased β-cell area and a decreased islet fibrosis, increased insulin secretion with increased GLP-1 secretion in response to a mixed meal, and an increased population of cells coexpressing GIP and GLP-1 in the jejunum anastomosed to the stomach. In contrast, DJB impaired glucose tolerance in nondiabetic Wistar rats. In conclusion, although DJB worsens glucose homeostasis in normal nondiabetic Wistar rats, it can prevent long-term aggravation of glucose homeostasis in diabetic GK rats in association with changes in intestinal enteroendocrine cell populations, increased GLP-1 production, and reduced β-cell deterioration.     

24-hour changes in circulating prolactin, follicle-stimulating hormone, luteinizing hormone, and testosterone in young male rats subjected to calorie restriction

This work analyzes the effect of calorie restriction on the 24 h variation of pituitary-testicular function in young male Wistar rats by measuring the circulating levels of prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Control animals were provided an equilibrium calorie diet and the experimental animals a calorie-restriction diet equivalent to 66% of food restriction for four weeks starting on day 35 of life. Different groups of control and experimental rats were killed at 6 h intervals around the clock, beginning 1 h after light on (HALO). Compared to the control animals, the mean secretion of prolactin was augmented and that of LH and testosterone decreased in calorie-restricted rats, whereas FSH release remained unchanged. Significant changes in the 24 h secretory pattern of circulating prolactin, LH, and testosterone occurred in the calorie-restricted rats. These include the appearance of a second maximum of plasma prolactin at 21 HALO, blunting of the LH peak seen at 13 HALO, and phase-shift of the testosterone peak from 13 HALO in controls to 17 HALO in calorie-restricted rats. The significant positive correlation between individual LH and testosterone levels found in controls was no longer observed in calorie-restricted rats. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of the pituitary-gonadal axis in growing male rats.     

Cell surface adenosine deaminase binds and stimulates plasminogen activation on 1-LN human prostate cancer cells

Adenosine deaminase (ADA) is expressed intracellularly by all cells, but in some tissues, it is also associated with the cell surface multifunctional glycoprotein CD26/dipeptidyl peptidase IV. By modulating extracellular adenosine, this "ecto-ADA" may regulate adenosine receptor signaling implicated in various cellular functions. CD26 is expressed on the surface of human prostate cancer 1-LN cells acting as a receptor for plasminogen (Pg). Since ADA and Pg bind to CD26 at distinct but nearby sites, we investigated a possible interaction between these two proteins on the surface of 1-LN cells. Human ADA binds to CD26 on the surface 1-LN cells and immobilized CD26 isolated from the same cells with similar affinity. In both cases, ADA binding is diminished by mutation of ADA residues known to interact with CD26. ADA was also found to bind Pg 2 in the absence of CD26 via the Pg kringle 4 (K4) domain. In the presence of 1-LN cells or immobilized CD26, exogenous ADA enhances conversion of Pg 2 to plasmin by 1-LN endogenous urinary plasminogen activator (u-PA), as well as by added tissue Pg Activator (t-PA), suggesting that ADA and Pg bind simultaneously to CD26 in a ternary complex that stimulates the Pg activation by its physiologic activators. Consistent with this, in melanoma A375 cells that bind Pg, but do not express CD26, the rate of Pg activation was not affected by ADA. Thus, ADA may be a factor regulating events in prostate cancer cells that occur when Pg binds to the cell surface and is activated.     

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4-hydroxylase of 1,2,3,4-tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean +/- SE) for hepatic microsomal TIQ 4-hydroxylase of male Wistar rats were 319.6 +/- 26.8 microM and 12.13 +/- 1.43 pmol.min-1.mg-1 protein, respectively. When TIQ 4-hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar greater than Dark Agouti) and sex (male greater than female) differences were observed. The microsomal activity toward TIQ 4-hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (beta-naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4-hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4-hydroxylase activity of 64.1 pmol.min-1.nmol P450(-1), which was 3.2-fold that of microsomes (20.9 pmol.min-1.nmol P450(-1)). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4-hydroxylase activity concentration-dependently. A male-specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol.min-1.nmol P450(-1)), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4-hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non-treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4-hydroxylation in rats pretreated with P448-inducers.     

全球森林生态系统碳储量、固碳能力估算及其区域特征

从气候地带性和地理区域分布两方面对森林生态系统碳储量及固碳能力,以及土地利用变化对森林固碳的影响和森林固碳估算不确定性的原因进行综述.据估算,全球森林生态系统碳储量为652～927 Pg C,固碳能力达到4.02 Pg C·a^-1.各气候地带森林碳储量表现为热带最大(471 Pg C),寒带次之(272 Pg C),温带(113～159 Pg C)最小,固碳能力表现为热带(1.02～1.3 Pg C·a^-1)最大,温带次之(0.8 Pg C·a^-1),寒带(0.5 Pg C·a^-1)最小;各地理区域森林碳储量表现为南美洲(187.7～290 Pg C)最大,其次是欧洲(162.6 Pg C)、北美洲(106.7 Pg C)、非洲(98.2 Pg C)和亚洲(74.5 Pg C),而大洋洲(21.7 Pg C)最小,固碳能力为南美洲热带(1276 Tg C·a^-1)和非洲热带(753 Tg C·a^-1)较大,其次是北美洲(248 Tg C·a^-1)和欧洲(239 Tg C·a^-1),而东亚(98.8～136.5 Tg C·a^-1)较小.为进一步减少森林生态系统固碳估算的不确定性,今后应综合运用连续长期观测技术、样地清查、遥感分析和模型模拟等方法.     

Plasminogen activation by antiplasminogen monoclonal antibody IV-1C. Properties and mechanism of reaction

In review the results of investigation of plasminogen(Pg) activation by antiplasminogen monoclonal antibody IV-1c have been presented. Antigenic determinant of IV-1c was localized in Val709-Gly718 site of Pg protease domain. IV-1c completely inhibited the Pg activation by streptokinase, but increased the rate of Pg activation by t-PA and urokinase. Catalytic properties of plasmin in complex with IV-1c were studied. It was found that IV-1c induced catalytic activity in Pg-IV-1c complex. It was shown that Pg and IV-1c interacts in complex by two-centre mechanism: IV-1c binds with Pg by paratope and by N-terminal lysine of gamma-chain and Pg binds to IV-1c by one of the lysine binding sites and by V709-G718 site of protease domain. The influence of pH, temperature, 1.5 mM Ca2+, Mg2+, Sr2+, Ba2+, Co2+, Ni2+ cations and 10 mM Cl-, F-, Ac-, SO4(2-), HPO4(2-) anions on lag and fast phases of Pg activation by VI-1c was investigated. It was revealed that Val709-Gly718 site was determining in Pg activation by IV-1c and streptokinase.     

Distribution of activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase in the cranial dura mater-arachnoid interface zone of the rat

Summary The distribution of the activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase was studied in the encephalic dura mater-arachnoid borderline (interface) zone of albino Wistar rats. Intense clustering of electron-dense granules that indicated alkaline phosphatase activity was observed in the inner dural cells, the neurothelial cells, the outermost row of the outer arachnoidal cells and in the intercellular cleft between the latter two (the so-called electron-dense band). The remainder of the outer arachnoidal cells contained almost no reaction product. Mg-adenosine triphosphatase activity was distributed differently; a lack of reaction product was observed not only in the outer arachnoidal cells, but also in the zone occupied by the electron-dense band. The data confirm histochemically the barrier properties of the dura mater-arachnoid interface zone.     

Experimental congenital toxoplasmosis in Wistar and Holtzman rats

Congenital toxoplasmosis was evaluated in Wistar and Holtzman rats using two strains of Toxoplasma gondii isolated in Brazil. Pregnant rats were inoculated by subcutaneous or intraperitoneal routes with 10(6) or 8 x 10(6) tachyzoites of N strain (virulent for mice) and by subcutaneous or oral routes with 10(2) or 1.2 x 10(3) cysts of P strain (avirulent for mice). The tissues of rat pups born from these rats were bioassayed for T. gondii infection. T. gondii was not observed in the pups born from rats inoculated with N strain. In the animals inoculated with P strain, congenital toxoplasmosis occurred in 22.8% (Wistar rats inoculated with 10(2) cysts by the subcutaneous route), 11.4% (Wistar rats inoculated with 10(2) cysts by the oral route), 21.2% (Wistar rats inoculated with 1.2 x 10(3) cysts by the oral route) and 2.9% of fetal infection (Holtzman rats inoculated with 10(2) cysts by the oral route). None of the pups born from chronically infected mother were infected with T. gondii.