Studies of calcitonin cells and parathyroid glands of the Indian palm squirrel, Funambulus pennanti in response to experimental hypercalcaemia.

Long term hypercalcaemia was induced in F. pennanti by alternate day intramuscular injections of 50,000 IU of vitamin D2 and by giving them 1% CaCl2 solution prepared in tap water to drink. The controls were not injected with vitamin D2 and were given tap water. The serum calcium levels at various stages of the experiment (1-29 days) show increased values as compared with those of control animals. The calcitonin cells in the treated animals generally exhibit an increase in their number up to the 15th day. Mitotic figures are also encountered between the 7th and the 15th day of treatment. This exhibits the increase in the number of C cells. Constant calcium challenge results in increased quantities of secretory granules among these cells up to the 15th day and in degranulation from the 17th day onwards. It also causes degenerative changes in a certain number of C cells. The parathyroids exhibit atrophic changes (25 days onwards) due to chronic hypercalcaemia. For short term hypercalcaemia, animals were injected intravenously with 1 ml of 10% solution of calcium gluconate. The calcitonin cells do not exhibit any change during the first half hour but thereafter they exhibit progressive degranulation, resulting in marked degranulation after 5 hours of the injection. The parathyroids remain unaffected throughout the experiment and show no histological change.     

Myometrial activity around estrus in sows: spontaneous activity and effects of estrogens,cloprostenol, seminal plasma and clenbuterol

A new, nonsurgical, open-end catheter technique was used to study spontaneous uterine activity around estrus in sows, and the effects of estrogens, seminal plasma, cloprostenol, and clenbuterol on uterine activity. In the first experiment, uterine activity was studied in 14 multiparous, cyclic sows, during one or more estrous cycles, from day -4 to day 4 of the cycle (day 0: first day of standing estrus). From a few days before estrus until estrus, the percentage of sows showing any uterine contractions increased from 55 to 100%, and frequency and mean amplitude of uterine contractions for these sows increased from 15 to 22/h, and from 20 to 40 mmHg on average. After estrus, uterine activity decreased. There were large differences between sows in uterine activity, which were consistent over the days of the cycle. In the second experiment, 11.5 microg of estrogens in 100 ml saline (n = 17), 100 ml seminal plasma (n = 5), 1 mg cloprostenol in 100 ml saline (n = 10), 0.30 mg clenbuterol in 100 ml saline (n = 11), or 100 ml saline (n = 5) was infused IU, after recording spontaneous activity. Infusion with saline or seminal plasma did not affect uterine activity. Estrogens increased frequency of contractions. Cloprostenol increased both frequency and amplitude of contractions. Clenbuterol reduced both frequency and amplitude of contractions. In conclusion, this study shows that spontaneous uterine activity in sows is increased around estrus, and it supports the role of estrogens in boar seminal plasma in affecting uterine activity around mating. Further, this study has yielded possible tools to study the relation between uterine activity and sperm transport.     

Ciprofloxacin concentrations in serum, bone and bone marrow of rabbits

Ciprofloxacin concentrations were determined in serum, bone and bone marrow of rabbits. Four experimental groups of animals were examined: group A (n = 6) received a dosage of 60 mg/kg/day intramuscularly for 4 weeks, groups B (n = 6), C (n = 15) and D (n = 15) received dosages of 120 mg/kg/day subcutaneously for 2 days, 2 weeks, and 4 weeks, respectively. In the kinetic portion of the study, peak serum concentrations of ciprofloxacin measured at the 15 min sampling time were: 2.61 +/- 0.27 micrograms/ml in the 60 mg/kg/day group (group A) and 3.24 +/- 0.78 micrograms/ml in the 120 mg/kg/day group (group B). At necropsy, rabbits in group A had mean ciprofloxacin concentrations of 3.60 +/- 2.27 micrograms/ml in serum, 2.24 +/- 1.19 micrograms/g in marrow and 1.19 +/- 0.44 micrograms/g in bone. Rabbits in group B achieved mean levels of 4.02 +/- 1.23 micrograms/ml in serum, 2.48 +/- 0.79 micrograms/g in marrow, and 1.35 +/- 0.40 micrograms/g in bone. Rabbits in group C achieved mean levels of 5.65 +/- 2.16 micrograms/ml in serum, 3.74 +/- 1.33 micrograms/g in marrow and 1.92 +/- 0.94 micrograms/g in bone. Rabbits in group D achieved mean levels of 7.24 +/- 2.50 micrograms/ml in serum, 4.48 +/- 1.68 micrograms/g in marrow, and 1.93 +/- 0.54 micrograms/g in bone. Differences between mean values for the four experimental groups were not statistically significant.     

Bioreactor systems in drug metabolism: synthesis of cytochrome P450-generated metabolites

In this communication, we report that suspension cultures of Sf21 insect cells, co-infected with baculovirus containing the cDNA for a single cytochrome P450 and NADPH-cytochrome P450 oxidoreductase, can be employed successfully as "bioreactors" for the synthesis of milligram quantities of cytochrome P450-generated metabolite(s). Three standard or probe substrates for the human P450s were chosen for the initial biosynthetic experiments: testosterone, diazepam, and diclofenac. Testosterone (100 microM, 2.88 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to 6beta-hydroxytestosterone (2.3 mg) and 15beta-hydroxytestosterone (0.18 mg). Diazepam (100 microM, 2.9 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to temazepam (1.1 mg), N-demethyldiazepam (0.35 mg), and oxazepam (0.15 mg). Diclofenac (100 microM, 3.18 mg/100 ml), added to a 100-ml CYP2C9 bioreactor, was converted to 4'-hydroxydiclofenac (2.6 mg). Since the goal for the development of the bioreactors was to provide a platform for both the production and subsequent purification of milligram quantities of P450-generated metabolite(s), a second 100-ml CYP2C9 bioreactor was used for the large-scale production and subsequent purification of 4'-hydroxydiclofenac. After 55 h of incubation, 7.95 mg of diclofenac was converted to 4.35 mg of 4'-hydroxydiclofenac, while 3.55 mg of unchanged diclofenac remained in the bioreactor. Using a simple preparative HPLC method, approximately 2.2 mg of 4'-hydroxydiclofenac and 1.9 mg of diclofenac were recovered from this experiment (28% yield). These results indicate clearly that suspension cultures of Sf21 insect cells coexpressing a cytochrome P450 and NADPH-cytochrome P450 oxidoreductase can be used effectively as bioreactors for the production and subsequent purification of milligram quantities of P450-derived metabolite(s).     

Effects of beta-carotene,vitamin C and E on antioxidant status in hyperlipidemic smokers

Smoking can accelerate the consumption of the stored antioxidant vitamins and increase the oxidative stress in the hyperlipidemic patients. The study investigated the effects of combined beta-carotene, vitamin C, and vitamin E on plasma antioxidant levels, erythrocyte antioxidative enzyme activities, and LDL lipid peroxides. Male hyperlipidemic smokers (35-78 years old) were randomly divided into two antioxidant supplemented groups: intervention 1 (I1, n = 22) (15 mg beta-carotene/day, 500 mg vitamin C/day, and 400 mg alpha-tocopherol equivalent/day) and intervention 2 (I2, n = 20) (30 mg beta-carotene/day, 1000 mg vitamin C/day, and 800 mg alpha-tocopherol equivalent/day). After 6-week supplementation, plasma beta-carotene, vitamin C, vitamin E, and erythrocyte glutathione levels increased significantly by 200%, 98%, 129%, and 39%, respectively, in the I1 group, and by 209%, 216%, 197%, and 32%, respectively, in the I2 group. Plasma Fe(+2) concentrations and Fe(+2)/Fe(+3) decreased significantly in both groups. Except erythrocyte glutathione peroxidase activity in the I1 group, erythrocyte catalase, glutathione peroxidase, and superoxide dismutase activities increased significantly in both groups. Lipid peroxides in LDL decreased significantly by 56% and 72% in the I1 and I2 groups, respectively. However, the levels of plasma iron, erythrocyte glutathione, and LDL lipid peroxides, and the activities of erythrocyte antioxidative enzymes did not differ between two groups. In conclusion, combined antioxidant supplements increased plasma antioxidant levels and antioxidative enzyme activities, and lowered LDL lipid peroxides in male hyperlipidemic smokers. Higher dosage of the supplements did not have an additive effect.     

Anti-cowpox virus activities of certain adenosine analogs, arabinofuranosyl nucleosides, and 2'-fluoro-arabinofuranosyl nucleosides

Nucleoside analogs were investigated for their potential to inhibit cowpox virus (a surrogate for variola and monkeypox viruses) in cell culture and in lethal respiratory infections in mice. Cell culture antiviral activity was determined by plaque reduction assays, with cytotoxicity determined by cell proliferation assays. Selectivity indices (SI's, 50% cytotoxic concentration divided by 50% virus-inhibitory concentration) were determined for 15 compounds. Three arabinofuranosyl (Ara) nucleosides showed activity in mouse mammary tumor (C127I) cells: guanine (Ara-G), thymine (Ara-T), and adenine (Ara-A) with SI's of 113, 61, and 95, respectively. The 2'-fluoro-Ara nucleosides of 5-F-cytosine (FIAC), 5-methyluracil (FMAU), and 5-iodouracil (FIAU) exhibited SI's of 148, 77, and 29, respectively. Other potent compounds included cidofovir (a positive control) and 3'-O-methyladenosine, with SI values of 164 and 56, respectively. In general, assays performed in African green monkey kidney (Vero) cells produced lower SI's than in C127I cells, except for 5-iodo-2'-deoxyuridine (IDU) which had an SI of > 71 in Vero cells and 3.1 in C127I cells. Intranasal infection of mice with cowpox virus was followed a day later by twice daily intraperitoneal treatment with compounds for 5 days. Ara-A was active at 300 mg/kg/day (40% survival), FMAU at 100 mg/kg/day (70% survival), and cidofovir (given for 1 day only) at 100 mg/kg (80-100% survival). None of the other compounds, including IDU, prevented death nor delayed the time to death. Cidofovir had the best potential for treating orthopoxvirus infections of those tested.     

2-12烷基-6-甲氧基环己基-2,5-二烯-1,4-二酮(DMDD)抗弥漫大B淋巴瘤的作用及机制*

目的:探讨2-12烷基-6-甲氧基环己基-2,5-二烯-1,4-二酮(DMDD)抗弥漫大B淋巴瘤(DLBCL)的作用及分子机制。方法:动物实验取4周龄BALB/C小鼠,分5组,20只/组,腹股沟注射DLBCL细胞株OCI-LY19细胞1 × 10⁷ cells/ml 每只0.1 ml,两天后分别灌胃0、1、5、25、125 mg/kg剂量的DMDD,1次/2天,给药的第18日,杀10只小鼠,取瘤组织称重,记录剩余小鼠的生存期。细胞实验取OCI-LY19细胞加入96孔培养板,每孔100 μl 1×10⁵ cells/ml,分别加入100 μl DMDD使其终浓度分别为0、1、5、25和125 μmol/L,作用0、24、48和72 h,设三复孔,MTS法检测细胞增殖活性;根据细胞增殖实验结果,选择0 μmol/L、5 μmol/L和25 μmol/L的DMDD作为后续用药浓度作用OCI-LY19细胞24 h,流式细胞仪分析凋亡率,hoechst染色观察细胞核型,JC-1染色观察线粒体膜电位,LDH释放实验评估药物细胞毒性,qPCR、Western blot分析基因转录和表达水平。结果:动物实验表明:与0 mg/kg用药组比,1～125 mg/kg DMDD能抑制小鼠瘤组织生长并延长其生存期(P＜0.01)。细胞实验表明:DMDD用药组OCI-LY19细胞增殖活性明显降低、凋亡水平显著增加(P＜0.01),细胞核出现碎裂、凝集和凋亡小体及线粒体膜电位下降,LDH释放率显著增加(P＜0.01),细胞内caspase-3和bax基因的转录表达和IκBα的磷酸化水平显著上调,bcl-2、bcl-xL、jak2和stat3基因的转录和蛋白表达水平明显受抑(P＜0.01)。结论:DMDD通过抑制JAK2/STAT3和NF-κB信号通路中JAK2、STAT3和p-IκBα的表达,下调BCL-2/BAX、活化Caspase-3,最终激活OCI-LY19细胞线粒体凋亡的内源性通路而促进了DLBCL细胞凋亡,抑制了OCI-LY19细胞增殖,具有抗DLBCL的作用。     

Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.     

Effect of Dissolved Oxygen, Temperature, Initial Cell Count, and Sugar Concentration on the Viability of Saccharomyces cerevisiae in Rapid Fermentations

By using 7 x 10(8) cells of Saccharomyces cerevisiae per ml with which 25 degrees Brix honey solutions were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C, i.e., rapid fermentation, the death rate was found to be high, with only 2.1% of the yeast cells surviving at the end of 3 h under anaerobic conditions. As the dissolved oxygen in the medium was increased from 0 to 13 to 20 to 100% in rapid fermentations at 30 C, there was a progressive increase in the percentage of cells surviving. The ethanol production rate and total were not seriously affected by a dissolved oxygen concentration of 13%, but fermentation was retarded by 20% dissolved oxygen and still further decreased as the dissolved oxygen content reached 100%. When the fermentation temperature was decreased to 15 C (at 13% dissolved oxygen), the rate of fermentation decreased, and the fermentation time to 9.5% ethanol (wt/vol) increased to 6 h. It was found that the higher the temperature between 15 and 30 C, the greater the rate of death as initial cell counts were increased from 1.1 x 10(7) to 7.8 x 10(8) cells per ml. At the lowest level of inoculum, 1.1 x 10(7) cells per ml, there was actual multiplication, even at 30 C; however, the fermentation was no longer rapid. The addition of 15% sugar, initially followed after an hour by the remaining 10%, or addition of the sugar in increments of 2.5 or 5% yielded a better survival rate of yeast cells than when the fermentation was initiated with 25% sugar.     

Oral administration of chitin down-regulates serum IgE levels and lung eosinophilia in the allergic mouse

Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1-10 micrometer polymers of N-acetyl-d -glucosamine) through mannose receptors and produce IL-12, IL-18, and TNF-alpha. These cytokines lead to the production of IFN-gamma by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 x 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of IL-4 (0.6 ng/ml), IL-5 (20 U/ml), and IL-10 (3.2 ng/ml), but not IFN-gamma. Ragweed/chitin stimulation resulted in significant decreases of IL-4, IL-5, and IL-10 levels and the production of IFN-gamma (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-gamma production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.     

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 by blood derived macrophages from a patient with alveolar rhabdomyosarcoma during short-term culture and 1 alpha,25-dihydroxyvitamin D3 after long-term culture

We have examined the ability of blood-derived monocytes and macrophages isolated from a patient with alveolar rhabdomyosarcoma and hypercalcaemia, to form 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) from 25-hydroxyvitamin D3 (25(OH)D3). Adherent monocyte-macrophage cells incubated with 25(OH)D3 over the initial 2 days in culture synthesized 1.9 pmol 24,25(OH)2D3/h/incubation (representing 0.63 pmol/h/10(6) cells), whereas macrophages synthesized 1.03 and 1.15 pmol 1 alpha,25(OH)2D3/h/incubation after 1 and 4 weeks in culture respectively. In a further experiment synthesis of 1 alpha,25(OH)2D3 by long-term cultured macrophages fell from 2.25 to 0.04 pmol/h/incubation following exposure to 10 nM 1 alpha,25(OH)2D3 for 7 days, whereas 24,25(OH)2D3 synthesis was induced (0.46 pmol/h/incubation). The vitamin D3 metabolites were identified by co-chromatography with authentic 24,25(OH)2D3 or 1 alpha,25(OH)2D3 in three different high-performance liquid chromatography systems. Serum 1 alpha,25(OH)2D3 in the patient was markedly suppressed at 5 pg/ml (normal 20-50 pg/ml) indicating that raised 1 alpha,25(OH)2D3 was not the cause of the hypercalcaemia, but rather, that raised calcium may have suppressed renal 1 alpha,25(OH)2D3 synthesis. Administration of APD (3-amino-1-hydroxypropylidine-1,1-bisphosphonate) corrected the hypercalcaemia in the patient suggesting that increased bone resorption was responsible for the raised calcium. The results of this study show for the first time that immature blood derived monocyte-macrophage cells can synthesize 24,25(OH)2D3 before they mature into macrophages able to synthesize 1 alpha,25(OH)2D3.     

Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit placental-conditioned medium stimulates progesterone accumulation by granulosa-lutein cells in culture: preliminary characterization of a placental luteotropic hormone

The rabbit fetal placenta plays an important physiological role in luteal maintenance in pregnancy, probably via the secretion of an unidentified placental "luteotropin." The objective of these studies was to examine conditioned medium from fetal placental-tissue incubations (FPI) for the presence of placental luteotropic hormones/factors, using the stimulation of progesterone accumulation by rabbit granulosa-lutein cells in culture, as an in vitro luteotropic bioassay. Progesterone accumulation by rabbit granulosa-lutein cells (during 5 days of culture) was increased (compared with controls), 1.5-fold by 10(-8) M estradiol-17 beta (E2) and 11.5-fold by 100 ng/ml luteinizing hormone (oLH). FPI stimulated progesterone accumulation (approximately 3-fold) and this was further increased in the presence of E2 (FPI + E2; approximately 6-fold). Luteotropic bioactivity in FPI (+ E2) was retained after dialysis (6000-8000 MW cutoff; 7.8-fold) and heating (90-95 degrees C for 1 h; 7.5-fold), but was destroyed after incubation with trypsin (1 mg/ml, 1 h at 37 degrees C; 0.9-fold). Media conditioned with skeletal muscle (1.2-fold), heart (1.6-fold), liver (1.5-fold), and uterus (0.5-fold) and 5-10% serum (less than 1-fold), from pseudopregnant rabbits, had little or no luteotropic bioactivity. These data indicate that FPI contains a luteotropic hormone/factor that is probably a heat-stable, trypsin-sensitive, protein/peptide of greater than 6000-8000 MW that acts in synergy with E2 to promote granulosa-lutein cell steroidogenesis. This placental hormone/factor is a good candidate for the elusive rabbit placental luteotropin.     

Enzymic activity of the K5-type yeast killer toxin and its characterization

K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25 mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in beta-glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg(+2), but increased with some other metal ions, most of all by Pb(+2).     

Alpha-tocopherol protects against monocyte Mac-1 (CD11b/CD18) expression and Mac-1-dependent adhesion to endothelial cells induced by oxidized low-density lipoprotein

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.     

The effect of stimulators and blockers of adrenergic receptors on LH secretion and cyclic nucleotide (cAMP and cGMP) production by porcine pituitary cells in vitro.

The direct effects of alpha- and beta-adrenergic agents on luteinizing hormone (LH) secretion in vitro by porcine pituitary cells and the participation of secondary messengers, adenosine 3'5'-monophosphate (cAMP) and guanosine 3'5'-monophospate (cGMP), in transduction of signals induced by adrenergic agents and gonadotropin-releasing hormone (GnRH) in these cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) 1 month before slaughter. OVX gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2 and 3) estradiol benzoate (EB; 2.5mg/100kg b.w.) at 30-36h (OVX+EB I) or 60-66h (OVX+EB II) before slaughter, respectively; (4) progesterone (P(4); 120mg/100kg b.w.) for 5 consecutive days before slaughter (OVX+P(4)). Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10(6) per well) in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days, at 37 degrees C and under the atmosphere of 95% air and 5% CO(2). On day 4 of the culture, the cells were submitted to 3.5h incubation in the presence of GnRH (a positive control), alpha- and beta-adrenergic agonists (phenylephrine (PHEN) and isoproterenol (ISOP), respectively), and alpha- and beta-adrenergic blockers (phentolamine (PHENT) and propranolol (PROP), respectively). The culture media were assayed for LH (experiment I) and cyclic nucleotides (experiment II).In experiment I, addition of GnRH (100ng/ml) increased LH secretion by pituitary cells taken from gilts of all experimental groups. The effects of alpha- and beta-adrenergic agents on LH secretion by the cells depended on hormonal status of gilts. The LH secretion by pituitary cells of OVX gilts was potentiated in the presence of PHEN (10, 100nM, and 1microM) and PHENT (1microM), alone or in combination with PHEN (100nM) and by the cells derived from OVX+EB I and OVX+P(4) animals in response to PHEN (100nM) and ISOP (1microM). ISOP (1microM) also stimulated LH secretion by the cells taken from OVX+EB II gilts. In experiment II, GnRH (100ng/ml) increased cGMP production by pituitary cells obtained from all groups of gilts and cAMP secretion by the cells taken from OVX and OVX+P(4) animals. PHEN (100nM) decreased and PROP (1microM) enhanced cAMP production by pituitary cells derived from OVX+EB I and OVX gilts, respectively. Moreover, PHEN (100nM) reduced, while PHENT (1microM) stimulated the release of cGMP by pituitary cells taken from OVX+EB II animals. In turn, ISOP (100nM) decreased and increased cGMP production by the cells derived from OVX+EB II and OVX+P(4) gilts, respectively. PROP (1microM) potentiated cGMP accumulation by pituitary cells taken from OVX+EB I and OVX+P(4) animals.In conclusion, our results suggest that adrenergic agents can modulate LH release by porcine pituitary cells acting through guanyl and adenylyl cyclase and in a manner dependent on hormonal status of gilts.     

Effects of sirolimus on plasma lipids,lipoprotein levels,and fatty acid metabolism in renal transplant patients

Sirolimus (Rapammune, rapamycin, RAPA) is a potent immunosuppressive drug that reduces renal transplant rejection. Hyperlipidemia is a significant side effect of sirolimus treatment, and frequently leads to cardiovascular disease. This study was undertaken to determine the repeatability, reversibility, and dose dependence of the plasma lipid and apolipoprotein altering effects of sirolimus, and to elucidate the mechanism by which sirolimus induces hypertriglyceridemia in some renal transplant patients. Six patients with renal allografts maintained on cyclosporine A and prednisone were selected on the basis of their previous hyperlipidemic response to short term (14 days) sirolimus administration. For longer-term treatment, each patient was started on 10 mg/day sirolimus and continued as tolerated for 42 days to reinduce hyperlipidemia. Timed blood samples were analyzed for lipid, apolipoprotein, and sirolimus levels. During sirolimus administration, mean total plasma cholesterol increased from 214 mg/dl to 322 mg/dl (+50%; range 25-92%); LDL-cholesterol levels followed a similar pattern. Mean triglyceride level rose from 227 to 432 mg/dl (+95%; range 9-254%). ApoB-100 concentration rose from 124 to 160 mg/dl (+28%; P < 0.05). ApoC-III level increased from 28.9 to 55.5 mg/dl, +92%; (P < 0.013). These lipid and apolipoprotein changes were found to be repeatable, reversible, and dose dependent. [(13)C(4)]palmitate metabolic studies in four patients with hypertriglyceridemia indicated that the free fatty acid pool was expanded by sirolimus treatment (mean = 42.3%). Incorporation of [(13)C(4)]palmitate into triglycerides of VLDL, IDL, and LDL was decreased 38.3%, 42,1%, and 38.4%, respectively, by sirolimus treatment of these patients. These results suggest that sirolimus alters the insulin signaling pathway so as to increase adipose tissue lipase activity and/or decrease lipoprotein lipase activity, resulting in increased hepatic synthesis of triglyceride, increased secretion of VLDL, and increased hypertriglyceridemia.     

Relationship among nitric oxide, leptin, ACTH, corticosterone, and IL-1beta, in the early and late phases of adjuvant arthritis in male Long Evans rats

Leptin, a hormone regulating body weight, food intake, and metabolism, is associated with activation of immune cells and inflammation. In this study we analyzed levels of leptin, adrenocorticotropic hormone (ACTH), corticosterone, interleukin 1beta (IL-1beta), and nitric oxide (NO) production on days 10 and 22 of adjuvant arthritis (AA) in male Long Evans rats to ascertain possible relationship of leptin with its modulators during the early and late phases of chronic inflammation. The circulating leptin levels were significantly reduced already on day 10 of AA compared to controls (1.97+/-0.22 ng/ml vs. 3.08+/-0.25 ng/ml, p<0.05); on day 22 no significant further drop was observed (1.06+/-0.21 ng/ml). Leptin mRNA in epididymal fat tissue was reduced in arthritic animals compared to controls on day 22 (0.61+/-0.09 vs. 1.30+/-0.1 arbU/GAPDH (p<0.01). IL-1beta concentration in spleen was enhanced on day 10 of AA (24.55+/-4.67 pg/100 microg protein vs. 14.33+/-1.71 pg/100 microg protein; p<0.05); on day 22 it did not differ from controls. ACTH and corticosterone levels were significantly elevated only on day 22 of AA (ACTH: 306.17+/-42.22 pg/ml vs. 157.61+/-23.94 pg/ml; p<0.05; corticosterone: 5.24+/-1.38 microg/100 ml vs. 1.05+/-0.23 microg/100 ml; p<0.01). Nitrate levels were enhanced similarly on days 10 (49.86+/-1.83 microM) and 22 of AA (43.58+/-2.17 microM), compared to controls (23.42+/-1.39 microM, p<0.001). These results show that corticosterone does not stimulate leptin production during AA. The suppression of leptin may be a consequence of permanent activation of NO, IL-1beta, and of lower weight gain. Circulating leptin does not seem to play a key role in the progression of chronic arthritis.     

Effect of aluminium hydroxide on serum ionised calcium,immunoreactive parathyroid hormone,and aluminium in chronic renal failure.

According to the Bricker-Slatopolsky theory, secretion of parathyroid hormone (PTH) is switched on in chronic renal failure by hypocalcaemia due to phosphate retention. In an attempt to reverse this process 20 patients in preterminal renal failure (plasma creatinine 569 +/- 195 mumol/l) were given aluminium hydroxide, 3.8 g daily. They were studied for four weeks and all measurements were made at the start and weekly, except measurements of serum aluminium concentration, which were made at the start and at the end of the fourth week. Mean serum phosphate fell from 1.89 to 1.47 mmol/l (5.9 to 4.6 mg/100), mean serum calcium rose from 2.07 to 2.24 mmol/l (8.3 to 9.0 mg/100 ml), and serum ionised calcium rose from 1.07 to 1.20 mmol/l (4.3 to 4.8 mg/100 ml), but serum immunoreactive PTH did not fall. Thirteen patients had initial serum immunoreactive PTH concentrations at or near to normal and 11 were taking beta-blockers but even in those with neither explanation, PTH concentrations did not fall. Serum aluminium concentrations rose from 0.4 to 1.02 mumol/l (10.9 to 27.4 microgram/l). Aluminium hydroxide corrects serum phosphate, total calcium, and ionised calcium at the price of a rise in serum aluminium concentration; in this study it did not affect serum immunoreactive PTH. The Bricker-Slatopolsky theory still needs verification in studies of patients with chronic renal failure.     

Ontogeny of tracheal fluid, pulmonary surfactant, and plasma corticoids in the fetal lamb.

We examined fetal plasma corticoids and flow rate, electrolyte composition, and surfactant content of tracheal fluid in chronic experiments with eight fetal lambs. From 120 to 148 days of gestation the rate of fluid production was 4.5 ml/kg per h, and there was no change in mean fluid sodium (147.8 meq/1), chloride (153.1 meq/1), calcium (2.2 mg/100 ml), and pH (6.23). Tracheal fluid potassium increased from 4.3 meq/1 at 120-130 days to 8.9 meq/1 at term, while plasma sodium, chloride, calcium, pH, and potassium were constant at 146.1 meq/1, 110.0 meq/1, 12.1 mg/100 ml, 7.39, and 4.0 meq/1, respectively. Plasma corticoids were less than 1.5 mug/100 ml total (0.3 mug/100 ml free) until 130 days, when they increased rapidly to 10.5 total (3.2 free) at 148 days. Surfactant was first detected in tracheal fluid between 124 and 133 days and its secretion increased rapidly after 135 days to a value of 125 mug/kg per h at 148 days. A sudden increase in fetal plasma corticoids does not seem to be the stimulus for appearance of surfactant in the lamb, although these hormones may induce the rapid accumulation of surfactant prior to delivery.