Meiosis in allopolyploid Crepis capillaris. II. Autotetraploids.

Meiotic chromosome pairing of autotetraploid Crepis capillaris was analysed by electron microscopy of surface-spread prophase I nuclei and compared with light microscopic observations of metaphase I chromosome configurations. Prophase I quadrivalent frequencies are high in all three tetrasomes. (A, D, and C) and partially dependent on chromosome size. At metaphase I quadrivalent frequencies are much lower and strongly dependent on chromosome size. There is no evidence for multivalent elimination during prophase I in this system, and the reduction in multivalent frequency at metaphase I can be explained by an insufficiency of appropriately placed chiasmata. The high frequencies of prophase I quadrivalents far exceed the two-thirds expected on a simple model with two terminal independent pairing initiation sites per tetrasome, suggesting that multiple pairing initiation occurs. Direct observations reveal relatively high frequencies of pairing partner switches (PPSs) at prophase I, which confirms this suggestion. The numbers of PPSs per tetrasome show a good fit to the Poisson distribution, and their positional distribution along chromosomes is random and nonlocalized. These observations favour a model of pairing initiation based on a large number of evenly distributed autonomous pairing sites each with a uniform and low probability of generating a PPS.

Eicosanoid levels in bronchoalveolar lavage fluid of young female smokers and non-smokers.

To evaluate indicators of inflammatory changes in the airways of young smokers we have measured the levels of several eicosanoids in bronchoalveolar lavage (BAL) fluid of 18 female smokers (age 33 +/- 2 years) and 9 female non-smokers (age 29 +/- 2 years) who were hospitalized for treatment not related to any pulmonary disease. In each BAL specimen the following eicosanoids were determined by radioimmunoassay: prostaglandin (PG) E2; PGF2 alpha; 9 alpha, 11 beta-PGF2, a metabolite of PGD2; 6-keto PGF1 alpha, a metabolite of prostacyclin; thromboxane (Tx) B2, a metabolite of TxA2; the 5-lipoxygenase products 5-hydroxy-eicosa-tetraenoic acid (HETE), leukotriene (LT) B4 and LTC4; the 12-lipoxygenase product 12-HETE; and the 15-lipoxygenase product 15-HETE. The concentrations of the cyclooxygenase products (pg ml-1) in the BAL fluid of the non-smokers were: PGE2 15.4 +/- 1.9, PGF2 alpha 7.6 +/- 1.0, 9 alpha, 11 beta-PGF2 8.7 +/- 1.8, TxB2 8.8 +/- 1.3, and 6-keto PGF1 alpha only 1.5 +/- 0.8. The concentration of the lipoxygenase products were: 15-HETE 781 +/- 200, 12-HETE 193 +/- 33, 5-HETE 14.0 +/- 3.1, LTC4 9.5 +/- 3.1, LTB4 6.2 +/- 1.4. BAL fluid from smokers contained two- to three-fold higher levels of TxB2 and PGF2 alpha (P less than 0.05). The levels of TxB2 and PGF2 alpha were positively correlated to the number of package years (rs = 0.55 and rs = 0.65, P less than 0.02). The concentrations of 5-, 12- and 15-HETE tended to be higher in BAL fluid from smokers, but this was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

15-HETE is the main eicosanoid formed by human colonic mucosa.

Eicosanoids were measured in colonic biopsies from eleven patients with active ulcerative colitis and from thirteen controls. Eicosanoid formation was measured after the addition of arachidonic acid and stimulation with calcium ionophore A23187. The 15-lipoxygenase derivative 15-hydroxy-eicosatetraenoic acid (15-HETE) was the predominant product formed in all biopsies. The amount of 15-HETE formed was dependent on the site of biopsy and decreased in the controls in biopsies taken in an aboral direction in the colon. The formation of 15-HETE and that of prostaglandin E2 (PGE2) and PGF2 alpha was proportional to the histologically obtained inflammation score. The role of 15-H(P)ETE as a mediator in ulcerative colitis should, therefore, be considered in addition to the effects of known modulators such as leukotriene B4 (LTB4) and PGE2.

The formation of thromboxane B2, leukotriene B4 and 12-hydroxyeicosatetraenoic acid by alveolar macrophages after activation during tumor growth in the rat.

Pieces of tumor tissue were implanted subcutaneously in the right flank of BN female rats. After 3, 7, 10, 12, 14 and 17 days the lungs were lavaged and the alveolar macrophages collected. The cells were activated with the calcium ionophore A23187 and the formation of thromboxane B2 (TxB2), leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12-HETE) determined. The formation of TxB2 decreased considerably until day 7. Thereafter, no changes occurred. The formation of LTB4 increased after the tumor implantation until day 10 and remained stable for the rest of the period, 12-HETE formation was approximately similar, with a decrease at day 12 but continued to increase after day 14. These results suggest that during tumor growth an inhibition of the cyclo-oxygenase or thromboxane synthase occurs and an activation of the C5- and C12-lipoxygenases of the alveolar macrophages.

Cellular and eicosanoid composition of bronchoalveolar lavage fluid in endotoxin protection against pulmonary oxygen toxicity.

Endotoxin protects against pulmonary oxygen toxicity in rats, and both prostaglandins and polymorphonuclear leukocytes (PMN) are implicated as playing an important role in this protective action. In this study, a bronchoalveolar lavage (BAL) technique was used to analyze cellular and eicosanoid composition of the lavage fluid of endotoxin-protected oxygen-exposed rats. The BAL fluid of the endotoxin-protected oxygen-exposed rats contained the highest number of PMN, while the BAL fluid of the nonprotected oxygen-exposed rats contained the highest number of macrophages. Thus, morbidity due to pulmonary oxygen toxicity was correlated with the number of macrophages but not with the number of PMN present in the BAL fluid. Leukotriene B4, thromboxane B2, and prostaglandin E2 levels were significantly higher in the lavage fluid of nonprotected oxygen-exposed rats compared to the levels in the lavage fluid of air-exposed rats. Eicosanoid levels in the BAL fluid of endotoxin-protected oxygen-exposed rats did not differ significantly from the levels found in air-exposed control rats. These findings suggest that endotoxin protects against hyperoxia-induced changes in eicosanoid metabolism.

Differential effects of malotilate on 5-, 12- and 15-lipoxygenase in human ascites cells.

Macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B4 and 5-monohydroxyeicosatetraenoic acid. The cyclooxygenase products 6-keto-PGF1 alpha and thromboxane B2 were the most important prostaglandin-like substances. Malotilate, an anti-fibrotic substance, selectively inhibited the 5-lipoxygenase, whereas both the 12- and the 15-lipoxygenase pathways were stimulated. The effects of malotilate on eicosanoid production differ from those of known lipoxygenase inhibitors. Such differential effects have not been reported previously.

Endotoxin protection against pulmonary oxygen toxicity and its reversal by acetyl salicylic acid: role of eicosanoid production by broncho-alveolar lavage cells.

Small doses of endotoxin markedly increase the survival rate of adult rats exposed to 98% oxygen for periods that are normally lethal. The lysine salt of acetyl salicylic acid (L-ASA) partially reverses this protective effect of endotoxin. In this pilot study we investigated the level of eicosanoid production by broncho-alveolar lavage (BAL) cells and found that BAL cells of endotoxin protected rats, present in abundance, have an equal or increased capacity of HHT, 15-HETE, 12-HETE, LTB4 and 5-HETE production. These data suggest that production of the lipoxygenase products by BAL cells does not seem to play an important role in the pathogenesis of pulmonary oxygen toxicity. We did not find any indication for the occurrence of shunting of arachidonic acid metabolism to the lipoxygenase pathway as an explanation for the reversal of endotoxin's protective action by L-ASA.

Determination of leukotriene B4, 6-keto-prostaglandin F1 alpha and thromboxane B2 in peritoneal lavage fluid of sham-operated and adrenalectomized rats.

In macrophages, isolated from the peritoneal fluid of rats, after activation, formation of metabolites of arachidonic acid occurs both by the cyclooxygenase and lipoxygenase pathways. The cells of normal animals produce mainly cyclooxygenase products. After adrenalectomy, a considerable increase occurs in the formation of lipoxygenase products, and less in those of the cyclooxygenase (1). In the experiments described here, the effect of adrenalectomy on the presence of leukotriene B4 (LTB4), 6-keto-PGF1 alpha and thromboxane B2 (TxB2) in the peritoneal fluid is determined.

Prostanoid imbalance in experimental acute necrotizing pancreatitis in rats.

In an investigation of the pathogenesis of acute necrotizing pancreatitis (ANP) the plasma levels of TXB2, 6-keto-PGF1 alpha, and PGE2 were measured in rats. After induction of ANP by injection of 5% sodium taurocholate into the pancreatic duct, a marked increase in TXB2 levels and a slight increase in 6-keto-PGF 1 alpha levels were found. PGE2 levels decreased. Mortality was 100% within 30 h. Pretreatment with chloroquine, a phospholipase A2 inhibitor, led to a inhibition of TXB2 production, whereas 6-keto-PGF1 alpha and PGE2 levels showed a surprising slight elevation in the first 6 h. Pretreatment with chloroquine decreased mortality by 30%. Pretreatment with FPL 55712, a leukotriene synthesis blocker, caused an increase in TXB2 and PGE2 levels, whereas the formation of 6-keto-PGF1 alpha remained unaltered. Two out of nine animals survived after pretreatment with FPL 55712. The results of the present study indicate that arachidonate end products are involved in ANP. The significance of the high TXB2 levels, decreased PGE2 levels, and only slightly elevated 6-keto-PGF1 alpha levels during ANP requires further investigation. The thromboxane A2 to prostacyclin ratio may be important.

Raised plasma thromboxane B2 levels in experimental acute necrotizing pancreatitis in rats. The effects of flunarizine, dazoxiben, and indomethacin.

The possible role of thromboxane A2 (TXA2) in acute necrotizing pancreatitis (ANP) was investigated in rats. After ANP was induced by injecting sodium taurocholate (5% w/v) into the pancreatic duct, the thromboxane B2 (TXB2) levels in plasma increased significantly. The effects of indomethacin, a general blocker of prostaglandin synthesis, on survival time and on plasma TXB2 levels were compared with those of dazoxiben, a more specific blocker of TXA2 synthesis, and Flunarizine, a calcium entry blocker known to inhibit the effects of TXA2. In a test group without any treatment, all animals died within 30 h of ANP induction. Although TXB2 levels were lowered by the administration of indomethacin, dazoxiben, and Flunarizine, survival times were not significantly altered. Indomethacin pretreatment had no beneficial effect, whereas 30% and 40% of the animals survived for 36 h after treatment with Flunarizine and dazoxiben, respectively. The results of the present study indicate that inhibition of TXA2 synthesis alone does not dramatically alter survival time. However, a potential role for other arachidonate metabolites in ANP cannot be ruled out by this study.

Comparison of the production of eicosanoids by human and rat peritoneal macrophages and rat Kupffer cells.

Human and rat peritoneal macrophages and rat Kupffer cells were labelled with [1-14C] arachidonic acid and stimulated with the calcium ionophore A23187. The metabolites formed were separated by high pressure liquid chromatography (HPLC). Human peritoneal macrophages formed especially leukotriene B4, 5-hydroxy-6,8,11,14 eicosatetraenoic acid and small amounts of leukotriene C4 and thromboxane B2, 12-hydroxy-5,8,10 heptadecatrienoic acid and 6-keto-prostaglandin F1 alpha, whereas rat peritoneal macrophages mainly produced cyclooxygenase products and in particular thromboxane B2 and 12-hydroxy-5,8,10 heptadecatrienoic acid. Rat Kupffer cells synthesized mainly cyclooxygenase products such as prostaglandin F2 alpha, prostaglandin D2 and prostaglandin E2. These results indicate that the profile of eicosanoids production by macrophages is dependent both on the species and on the tissue from which the macrophage is derived.

The effect of adrenalectomy on the aggregation of rat blood platelets and the endogenous formation of thromboxane B2 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid.

Rat platelets were isolated and labelled with [1-14C] arachidonic acid. After aggregation thromboxane B2, 12-hydroxy 5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-eicosatetraenoic acid (12-HETE) were the main metabolites formed. A comparison was made between several properties of the platelets of adrenalectomized and sham operated rats. There was no difference in collagen-induced aggregation. The amount of 12-HETE and the sum of TxB2 and 12-HETE formed from endogenous arachidonic acid after aggregation was higher in the first group.

Formation of prostaglandins and leukotrienes by human lung tissue in vitro after activation by the calcium ionophore A23187.

The formation of metabolites of arachidonic acid by the cyclo-oxygenase and lipoxygenase pathways were determined in human lung tissue, obtained from surgery. In this measurement the chopped tissue was incubated with the calcium ionophore A23187. Formation of metabolites from [1-14C] arachidonic acid was also determined. The metabolites were extracted, separated by HPLC and identified by measurement of the absorption spectrum at 280 nm, radioactivity, biological activity and by radioimmunoassay. 6-keto-prostaglandin F1 alpha (6-ketoPGF1 alpha), the metabolite of prostacyclin, is the cyclo-oxygenase product present in the highest amount (400 +/- 49 ng g-1), followed by PGD2 (162 +/- 59 ng g-1) thromboxane B2 (102 +/- 32 ng g-1) PGE2 (104 +/- 46 ng g-1) and PGF2 alpha (58 +/- 26 ng g-1). The amounts of the lipoxygenase products are: leukotriene B4 (LTB4), 163 +/- 100 ng g-1; LTC4, 63 +/- 31 ng g-1 and LTE4 121 +/- 34 ng g-1. From [1-14C] arachidonic acid higher amounts of the cyclooxygenase than of the lipoxygenase products were formed, with the exception of PGE2. The effects of several of these substances on the contraction of human small airway smooth muscle were measured. The contractions, induced by equivalent amounts of LTC4 and a synthetic analogue of thromboxane T X A2 were approximately one hundred times those induced by PGD2, PGF2 alpha and histamine. These results suggest that thromboxane A2 and LTC4 are the most important arachidonic acid metabolites that induce bronchoconstriction in the human lung.

Formation of leukotriene B4, 20-hydroxy leukotriene B4 and other arachidonic acid metabolites by macrophages during peritonitis in patients with continuous ambulatory peritoneal dialysis.

Macrophages, isolated from dialysis fluid of three patients with continuous ambulatory peritoneal dialysis (CAPD) at different times during peritonitis were labelled with 14C-arachidonic acid and stimulated with the calcium ionophore A23187. The main metabolites formed by 5-lipoxygenase activity were leukotriene B4 (LTB4) and 5-hydroxy-6, 9, 11, 14-eicosatetraenoic acid (5-HETE). Smaller amounts of cyclooxygenase metabolites were present and also a major compound with an elution time between 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TxB2). This substance was isolated, analyzed by GC-MS and identified as 20-hydroxy-leukotriene B4 (20-OH-LTB4). This indicates that human peritoneal macrophages obtained from CAPD not only produce leukotrienes and prostaglandins, but also the omega-hydroxylase product of LTB4, which has been demonstrated to be present in polymorphonuclear leucocytes. The activity of this enzyme was not correlated with the severity of the peritonitis.

Increase in the formation of leukotriene B4 and other lipoxygenase products in peritoneal macrophages of adrenalectomized rats.

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12-HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals.

Increase in the formation of cyclooxygenase and lipoxygenase products in kidneys of adrenalectomized rats.

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products from [1-14C]-arachidonic acid in rat kidneys after incubation with the Calcium-ionophore A23187 has been determined. The metabolites were isolated by HPLC. The main components formed are PGD2, PGE2, PGF2 alpha, LTB4, 5,12 di-HETE and 5-, 12- and 15-HETE. After adrenalectomy, an increase occurs in the formation of PG and LT, which is highest in that of PGD2 and 12-HETE. These effects are most probably related to a diminished formation or inactivation of lipocortin in adrenalectomized animals, a glucocorticosteroid induced peptide with phospholipase A2 inhibitory activity.