Cartilage growth inhibition and necrosis in vitro caused by prostaglandin A

This paper details experiments using the Fell technique of organ culture of 8-day chick embryo femoral and tibial rudiments to test the effects of prostaglandin A1 (PGA1) on cartilage growth. Growth was studied during 8 days in vitro by measurement of rudiment length and wet and dry weight, and by histology. PGA1 inhibited explant growth in a dose-related manner. Linear growth was significantly decreased by 20 and 25 microgram/ml PGA1 at 2, 4, 6 and 8 days, and by 15 microgram/ml at 6 and 8 days. Linear growth was unaffected by 1 and 10 microgram/ml doses. Weight measurements were significantly reduced by 25 microgram/ml PGA1 (2, 4 and 8 days) and by 20 microgram/ml (8 days). Chondroblast degeneration was caused by doses of 15, 20 and 25 microgram/ml PGA1. Progressive degeneration was seen at the 25 microgram/ml concentration after 2 days in vitro. Cellular changes as early as 27 h in vitro were seen using electron microscopy. Tritiated thymidine autoradiography confirmed reduced chondroblast proliferation in the presence of PGA1 (25 microgram/ml). The mechanisms of prostaglandin-induced changes in embryonic cartilage remain uncertain. The possible role of intracellular cyclic nucleotides in the reaction is discussed.     

Evidence for repair of ultraviolet-induced damage in Cystobacter species (Myxobacterales).

Cystobacter species strain CK 1 does not grow with more than 0.2 microgram/ml acriflavine. Spontaneous two-step mutants growing with 2 microgram acriflavine per ml have been selected. One mutant (strain CK3) was used to investigate the effect of repair inhibitors. Both strains exhibit pronounced shoulders in their UV dose curves of inactivation. Acriflavine (AF), coumarin (CU), and caffeine (CA) when incorporated in the post-irradiation plating medium decreased survival of irradiated cells. Post-treatment with 2 microgram acriflavine/ml abolished the shoulder of the curve. Caffeine (1600 microgram/ml) and coumarin (350 microgram/ml) reduced it only to about 40%. It is concluded that probably two repair mechanisms are present. Pre-treatment of the cells with 2 microgram acriflavine/ml for two hours before UV-irradiation resulted in a constant dose enhancement factor of 1.9. The protective effect is increased with the time of treatment with acriflavine. This may indicate that pyrimidine dimers are responsible for UV-inactivation.     

Pulsatile secretion pattern of growth hormone during the luteal phase and mid-anoestrus in beagle bitches

The pulsatile secretion pattern of growth hormone was investigated during four stages of the luteal phase and during mid-anoestrus in six cyclic beagle bitches. Plasma samples were obtained via jugular venepuncture at 10 min intervals for 12 h at 19 +/- 2 (mean +/- SEM; luteal phase 1), 38 +/- 2 (luteal phase 2), 57 +/- 2 (luteal phase 3), 78 +/- 2 (luteal phase 4) and 142 +/- 4 days (mid-anoestrus) after ovulation. During all stages, growth hormone was secreted in a pulsatile fashion. The mean basal plasma growth hormone concentration during luteal phase 1 (2.2 +/- 0.3 microgram l(-1)) was significantly higher than that during luteal phase 4 (1.5 +/- 0.1 microgram l(-1)) and mid-anoestrus (1.4 +/- 0.2 microgram l(-1)). The mean area under the curve (AUC) above zero during luteal phase 1 (27.3 +/- 2.7 microgram l(-1) in 12 h) tended to be higher than that during luteal phase 4 (20.8 +/- 1.8 microgram l(-1) in 12 h) and mid-anoestrus (19.2 +/- 2.5 microgram l(-1) in 12 h). In contrast, the mean AUCs above the baseline during luteal phase 1 (1.1 +/- 0.5 microgram l(-1) in 12 h) and luteal phase 2 (1.2 +/- 0.5 microgram l(-1) in 12 h) were significantly lower than that during luteal phase 4 (2.8 +/- 0.5 microgram l(-1) in 12 h). In conclusion, the pulsatile secretion pattern of growth hormone changes during the luteal phase in healthy cyclic bitches: basal growth hormone secretion is higher and less growth hormone is secreted in pulses during stages in which the plasma progesterone concentration is high. It is hypothesized that this change is caused by a partial suppression of pituitary growth hormone release by progesterone-induced growth hormone production in the mammary gland. The progesterone-induced production of growth hormone in the mammary gland may promote the physiological proliferation and differentiation of mammary gland tissue during the luteal phase of the bitch by local autocrine-paracrine effects. In addition, progesterone-induced mammary growth hormone production may exert endocrine effects, such as hyperplastic changes in the uterine epithelium and insulin resistance.     

Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages.

Macrophages isolated from the peritoneal cavity of untreated mice and maintained in tissue culture synthesize and release prostaglandins when challenged with zymosan. These cells also selectively release lysosomal acid hydrolases under the same conditions. The major prostaglandins released into the media are found to be prostaglandins E1, E2 and 6-oxoprostaglandin F1a, whereas prostaglandin F2a is not detected. Macrophages isolated from mice that have received an intraperitoneal injection of thioglycollate broth are far less responsive to zymosan challenge. These cells require 300 microgram of zymosan to synthesize and release one-third the amount of prostaglandins released from non-stimulated macrophages exposed to 50 microgram of zymosan. In addition, thioglycollate-stimulated macrophages release less than 10% of their lysosomal acid hydrolases when exposed to 300 microgram of zymosan whereas non-stimulated cells release approximately 50% of these enzymes after treatment with 50 microgram of zymosan. The zymosan-stimulated synthesis and release of prostaglandins are completely inhibited by indomethacin, whereas the increased selective release of lysosomal acid hydrolases is not affected. Macrophages, unlike fibroblasts, do not synthesize and release prostaglandins when exposed to serum or to bradykinin.     

The metabolism in man of (3H)-5alpha-16-androsten-3alpha-ol and of (3H)-5alpha-16-androsten-3-one

The in vitro metabolism in man of two 16-androstene steroids, 5alpha-16-androsten-3-one and 5alpha-16-androsten-3alpha-ol, has been studied using 3H-labelled tracers. 4 healthy subjects (2 of each sex) were chosen, and each labelled steroid was administered, by a single injection, to 1 man and 1 woman. Disappearance of (3H)-3alpha-androstenol in the subjects receiving this compound followed a curve which indicated a two-pool distribution in both cases; metabolic clearance rates for these subjects were found to be 3,790 1/24 h in the man and 3,120 1/24 h in the woman. Blood production rates calculated for the 3alpha-androstenol-treated subjects were 875 microgram/24 h in the man and 1,780 microgram/24 h in the woman. Recovery of 3H in the urine of all 4 subjects was very low, between 28 and 42%. Conversion of the injected precursors to urinary 3alpha-androstenol was 13.5 and 12.7% in the 2 men and 6.1 and 5.9% in the 2 women. The male subjects were found to have a lower 24-hour urinary 3alpha-androstenol output (570 and 387 microgram/24 h) than the average for men of their age. The urinary 3alpha-androstenol output in the women was 225 and 276 microgram/24 h, and was within the normal range for women. The urine production rates of 3alpha-androstenol were 2,470 and 4,090 microgram/24 h in the male and female subjects, respectively; the difference between the blood and urine production rates of this compound are thought to indicate the direct secretion of conjugates. Urine production rates of 5alpha-androstenone (measured as 3alpha-androstenol) were 2,370 and 4,340 microgram/24 h in the male and female subjects, respectively.     

A possible involvement of adrenaline in the facilitation of lordosis behavior in the ovariectomized rat

In order to examine a possible role of adrenaline (AD) or noradrenaline (NA) in the control of lordosis behavior, lordosis quotient (LQ) was observed daily for 8 consecutive days in the ovariectomized rat given daily 1 or 2 microgram/0.1 ml oil of estradiol benzoate (EB) alone or together with 100 microgram/0.1 ml saline of AD or NA. AD but not NA treated together with EB caused a greater change in the daily LQ than the same dose of EB alone and the change in the daily LQ by daily treatment with both 1 microgram EB and 100 microgram AD was equivalent to that by daily treatment with 2 microgram EB alone. A half mg progesterone (P) could induce the lordosis behavior in the ovariectomized rat treated 48 hr prior with both 1 microgram EB and 50 or 100 microgram AD, but not in the one treated with 1 microgram EB alone. While 50, 100 or 200 microgram NA or 10 microgram AD had no effect, 50 or 100 microgram AD pretreated together with 2 microgram EB produced a markedly higher LQ after P than 2 microgram EB alone in the ovariectomized rat. This effect of AD on the induction of lordosis behavior was produced only when AD was pretreated simultaneously with EB and AD priming 24 or 43 hr after EB failed to elicit the effect. Therefore, it is suggested that a change of the brain target site in the estrogen sensitivity produced by AD plays a part in the control of lordosis behavior.     

Gelatin of Limulus amoebocyte lysate by simple polysaccharides

The Limulus lysate gelatin activity of several simple polysaccharides including yeast mannans and bacterial dextrans was investigated. The mannans from Saccharomyces cerevisiae wild and mutant strains possessing dense branches showed positive gelatin activity at concentrations of 1 microgram/ml or more regardless of differences in their chemical structure. However, two synthetic mannans possessing linear structures with alpha 1 leads to 2 and alpha 1 leads to 6 linkages also gave positive reactions at concentrations of 10 microgram/ml or more and 500 microgram/ml or more, respectively. The dextran from Leuconostoc mesenteroides IAM 1046 consisting of a dense branching moiety displayed reactivity at concentrations of 100 microgram/ml or more, while the dextrans devoid of such branches were negative in this reaction. The optimal concentration for Limulus lysate gelatin could not be determined for any of the polysaccharides and lipopolysaccharides (LPS) tested in this study. The gelation activity of the polysaccharides was stable to treatment with 100 mM NaOH at 30 C for 72 hr. The minimum concentration for the gelation activity of LPS treated with 100 mM NaOH under the same conditions was reduced from 10(-6) approximately(-9) microgram/ml to 1-10 microgram/ml. The above findings demonstrate that the major part of Limulus lysate gelation activity of LPS depends on the alkali-degradable lipid A moiety, and that such simple polysaccharides are also able to participate in this activity even though the extent of participation is very low.     

Metabolic response to short periods of starvation in hypo- and hyper-thyroid male rats.

1) Thyroidectomized rats were fed with a low iodine diet, injected daily with 0, 0.1, 1.8 or 25 microgram of L-thyroxine/100 g body wt., and compared with intact controls. 2) Plasma protein-bound iodine was decreased in the rats given the 0 and 0.1 microgram doses, unchanged in those given the 1.8 microgram doses, unchanged in those given the 1.8 microgram dose increased in those given the 25 microgram one. 3) The liver content of DNA-P, phospholipid-P, proteins and fatty acids was decreased in the rats that did not receive thyroxine, practically recuperated in those receiving 0.1 microgram and normal in those given 1.8 or 25 microgram of thyroxine. 4) 3 h of starvation produced a reduction in the liver content of total fatty acids that disappeared after 24 h. 5) When fed, liver glycogen concentration was low in the rats given 25 microgram of thyroxine. 6) With starvation, the fall in liver glycogen and blood glucose, and the rise in liver acetyl-CoA and citrate and blood glycerol concentrations were faster in the thyroidectomized rats that did not receive thyroxine than in the other groups. 7) The rise in plasma free fatty acid and blood ketone bodies concentrations were similar in all the groups, the greater level of the first parameter being observed after 6 h of starvation in the rats given 25 microgram of thyroxine and in the second one after 24 h in the rats given either 0.1, 1.8 or 25 microgram of thyroxine. 8) The rapid decrease in the availability of carbohydrate stores with starvation in the thyroidectomized rats could be responsible for their fast call for lipid utilization. The slower response to fasting in the hyperthyroid animals is probably a consequence of their reduced amount of endogenous substrates to be mobilized.     

Effect of dexamethasone on the expression of p34(cdc2) and cyclin B1 in pig oocytes in vitro

The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition. At the G2-M boundary, maturation promoting factor (MPF) activation is usually induced via several ways, including tyrosine dephosphorylation of p34(cdc2) and synthesis of cyclin B according to cell type and species. Previous studies in our laboratory demonstrated that glucocorticoids directly inhibit the meiotic maturation of pig oocytes in vitro. The aim of this study was therefore to investigate the influence of glucocorticoids on the expression of p34(cdc2) and cyclin B1 in resumption of meiosis of pig oocytes. We detected the relative levels and association of p34(cdc2) and cyclin B1. Isolated cumulus-enclosed oocytes were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microgram/ml), LH (0.5 microgram/ml), FSH (0.5 microgram/ml), and estradiol-17beta (1 microgram/ml) in the presence or absence of dexamethasone (DEX) for 24 hr; they then were cultured without hormonal supplements in the presence or absence of DEX for an additional 24 hr. We found that cyclin B1, as well as p34(cdc2), was already present in fully grown G2-arrested pig oocytes when removed from the follicle. In these oocytes, cyclin B1 and p34(cdc2) were already associated in complex. Treatment with DEX at concentrations of 1 microgram/ml or above decreased the level of cyclin B1, but had no effect on the level of p34(cdc2). The exposure of oocytes to DEX also decreased the amount of complexed p34(cdc2)-cyclin B1. These findings suggest that the inhibitory action of DEX on meiotic maturation could be due, at least in part, to the reduced amount of p34(cdc2)-cyclin B1 complex.     

Nifedipine and verapamil block the memory-facilitating effect of corticotropin-releasing factor in rats

The effects of two calcium channel blockers, nifedipine and verapamil, and the peptide corticotropin releasing factor (CRF) as well as their interactive effects on memory retention were examined in rats. Male Sprague-Dawley rats were chronically cannulated bilaterally and drugs were directly injected into the dentate gyrus of the hippocampus. Animals were trained in a one-way inhibitory avoidance task and memory was measured 24 h later. Results indicate that there was a U-shaped dose-response curve for the effects of nifedipine and verapamil with nifedipine at 8 micrograms and verapamil at 1 microgram both impaired memory formation, while CRF at 0.1 microgram enhanced this process. Nifedipine at 2 micrograms and verapamil at 0.5 microgram, which did not have significant effects on memory by themselves, antagonized the memory-enhancing effect of CRF in the hippocampus. These results suggest that under normal physiological conditions, calcium influx may play an important role in memory consolidation process in the vertebrate.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B2 (TXB2) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 microgram/g tissue) to both PGE2 (10.7 microgram/g tissue) and TXB2 (6.2 microgram/g tissue. Smaller amounts of PGF2alpha (0.9 microgram/g) and 6-oxoPGF1alpha were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE2, but formed very little TXB2, PGF2alpha or 6-oxoPGF1alpha. Homogenates from rabbit lungs converted arachidonic acid (100 microgram/g) mainly to PGE2, both before and after birth. The amount of PGE2 formed increased during gestation to a maximum of about 6 microgram/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 microgram/g) which was observed 8 days after birth, followed by an increase to about 4 microgram/g in older rabbits.     

Increase in the activity of an epileptogenic focus in frog hippocampus as a result of kynurenines and its reduction by serotonin]

In frogs with epileptogenic focus induced by injection of penicillin (1000 U in 0.4 microliter) into the primordial hippocampus it was shown that pretreatment with two kynurenines (quinolinic acid -- 0.1 microgram, and d,l-kynurenine -- 1 microgram) into the focus region and their injection into the functioning epileptogenic focus led to a sharp increase of the interparoxysmal epileptiform discharges and electrographic correlates of the fit on the EEG. Anthranilic acid (5 microgram) did not influence the activity of epileptogenic foci, and serotonin (1 microgram) and 5-methoxytryptamine (1 microgram) decreased it significantly. It is suggested that the effect of kynurenines on neurones in the epileptogenic foci may play a certain role in the pathogenesis of epilepsy.     

Screening and identification of antimicrobial compounds from Streptomyces bottropensis suppressing rice bacterial blight

Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating pathogen to Oryza sativa and has been shown to cause bacterial blight. Two bioactive compounds showing antimicrobial activities against Xoo strain KACC 10331 were isolated from a Streptomyces bottropensis strain. The ethyl acetate extract was fractionated on a Sephadex LH-20 column, and then purified by preparative HPLC. The purified compounds were identified as bottromycin A2 and dunaimycin D3S by HR/MS and 1H NMR analyses. The MIC value against Xoo and the lowest concentration still capable of suppressing rice bacterial blight were 2 microgram/ml and 16 microgram/ml for bottromycin A2, and 64 microgram/ml and 0.06 microgram/ml for dunaimycin D3S, respectively. These two compounds were shown to exert different bioactivities in vitro and in rice leaf explants.     

Isolation and characteristics of polymyxin-resistant mutants of Shigella flexneri

Some characteristics of S. flexneri 2a mutants resistant to various concentrations of polymyxin M were studied. The data indicate that mutations resulting in low (50 microgram/ml) and high (300 microgram/ml) levels of the antibiotic resistance were determined by different genes. Polymyxin resistance led to changed permeability of the outer membrane with respect to detergents and some antibiotics, such as aminoglycosides, penicillins, chloramphenicol and amphotericin B but did not change sensitivity of the strains to some bacteriophages, except phage PI. Mutants resistant to 50 microgram/ml of polymyxin M preserved their ability to induce keratoconjunctivitis in guinea pigs. Part of the strains resistant to 300 microgram/ml of the antibiotic lost this property. No correlation between the polymyxin M resistance level, loss of the pathogenic properties and toxicity of the bacterial cells was found. It was confirmed that though inactivation of endotoxin by polymyxins is associated with their capacity for interaction with lipid A, this component does not participate in development of resistance to these antibiotics.     

Effects of copper on growth, reproduction, survival and haemoglobin in Daphnia magna

The effects of additions of CuSO4 X 5H2O to final concentrations between 0.0004 and 105 micrograms Cu l-1 on growth, reproduction, survival and haemoglobin content of Daphnia magna were studied in hard reconstituted water and compared to the response in the dilution water without addition of copper. Concentrations of copper are nominal values. The 48-hr EC50 (immobilization) for unfed neonates was 6.5 micrograms Cu l-1 and the 48-hr and 21-day LC50 for fed neonates were 18.5 and 1.4 microgram Cu l-1, respectively. Growth expressed as body length of juveniles after 7 days and adult females after 21 days was only reduced in survivors at the highest non-lethal concentration (6.6 micrograms Cu l-1). Reproduction was stimulated by low concentrations of copper. Optimal reproduction after 21 days was found between 0.001 and 0.1 microgram Cu l-1. Higher concentrations were partially inhibitory (0.4 microgram Cu l-1), stimulatory (0.8 and 1.6 microgram Cu l-1) or completely inhibitory (3.2 micrograms Cu l-1 and above). The stimulatory peak around 1 microgram Cu l-1 was accompanied by a reduced survival (above 0.4 microgram Cu l-1). The Zero Equivalent Point (ZEP) for reproduction at non-reduced survival was 0.23 microgram Cu l-1. This concentration should be "safe" for D. magna under prevailing conditions (reconstituted water with a hardness of 250 mg l-1 as CaCo3 and a synthetic diet based on fish food and baby gruel). The haemoglobin content was affected by copper in a complex pattern which was not related to growth, reproduction or survival.     

A comparison of grooming behavior potencies of neurohypophyseal nonapeptides

We have previously demonstrated that intracerebroventricular (ICV) administration of oxytocin (OXY) enhanced grooming behaviors in male and female rats at a 1 microgram dose. In the present study female rats were injected ICV with 1 microgram OXY or equimolar doses of other peptides. At this dose arginine-vasopressin (AVP), arginine-vasotocin (AVT) and lysine-vasopressin (LVP), as well as alpha-MSH, were as effective as OXY in increasing grooming behavior. At equimolar doses, ACTH1-10, tocinoic acid (the ring structure of OXY) and Pro-Leu-Gly-NH2 (the tail structure of OXY) had no significant effect on grooming behavior. The potency of AVP and AVT was determined across a 0.05-5 microgram dose range. Grooming scores increased in an apparent linear manner across a similar OXY dose range. Both AVP and AVT, however, manifested an inverted U grooming response curve. Maximum grooming scores resulted from a 0.1 microgram dose of AVT or a 0.5 microgram AVP dose. Analyses of the aspects of grooming separately found that nonapeptides OXY, AVP and AVT all elevated body grooming, washing, and scratching. Because AVT and AVP administration resulted in grooming scores significantly higher than OXY at lower doses, we concluded that the CNS is more sensitive to the effects of AVT and AVP on grooming behavior than OXY.     

The relation between the synergistic effect of cytostatics and metabolism of V 79B cells in vitro.

A single-dose simultaneous application of methotrexate (MTX; 0.002/microgram ml-1) and cisplatin (cis-Pt; 0.0002/microgram ml-1) had a permanent synergistic effect on both synchronized and asynchronous cell populations of V 79B cells. Successive combination of the drugs was manifested synergistically when MTX was applied first. The synchronized cell population was more sensitive to the cytostatics than the asynchronous population. Treatment with MTX alone, or the combination of MTX-cis-Pt, as well as their successive combination with the first drug being cis-Pt, caused gluconeogenesis.     

A time course study of the effect of repetitive doses of estradiol-17 beta on serum glucose and lipids, liver glycogen and some carbohydrate metabolizing enzymes in liver of male flounder (Platichtys flesus L.)

1. Repeated treatment of male flounder with 5 and 100 microgram doses of estradiol-17 beta increases the level of phospholipid, triglyceride, free fatty acids and total lipid in serum as a function of time and dose, during a period of 17 days. 2. The glucose level in serum is increased and decreased respectively by doses of 5 and 100 micrograms. 3. Five and 100 microgram doses decrease the level of glycogen in liver. 4. Five microgram doses do not affect the activity of the measured enzymes considerably, with the exception of phosphorylase a. 5. One hundred microgram doses increase the activity of glucose-6-phosphate dehydrogenase after 11 days. 6. One hundred microgram doses increase the activity of pyruvate kinase continuously during the experimental period and decrease phosphorylase a and glucose-6-phosphatase activity.     

Action of an antiestrogen agent, tamoxifen on the uterus and vagina of the ovariectomized rat]

Tamoxifen has more or less strong estrogen influence according to the targets : a light one on the uterus (1 mg being much less strong than 0.25 microgram of estradiol), a dynamic one on the vagina (50 microgram of tamoxifen make the vagina open in as short a time as 0.25 microgram of estradiol do but the keratinisation is still not completed even with 1 mg of tamoxifen). We can still see this influence four days after the end of the treatment. This influence is weak on the uterus until the 11 th day and it is much stronger on the vagina until about the 8 th day. Tamoxifen has an antiestrogenic action when opposed to 20 microgram of estradiol : this action is limited as soon as you give a dosis of 50 microgram on the uterus and it is nearly total with a dose of 1 mg ; we can notice it on the vaginal only from 200 microgram on.