Effects of insulin, somatomedin--a multiplication stimulating activity (MSA)--and transferrin, on the lipolysis of rat adipocytes in vitro

Antilipolytic effect was researched when insulin (0.1 and 1 mIU/ml), MSA (200 and 500 ng/ml) and transferrin (2 and 5 micrograms/ml) were added to a suspension of freshly isolated rat adipocytes in vitro. Lipolysis was measured as glycerol secretion in the medium: micromoles/90 minutes/100 mg total lipids. Insulin (1 mIU/ml) reduced adrenalinic stimulation of lipolysis: A 1 microgram/ml (P less than 0.05). MSA 200 ng/ml had no effect. MSA 500 ng/ml reduced basal lipolysis and adrenalinic stimulation (P less than 0.05), and increased insulin-induced antilipolysis (P less than 0.05). Transferrin was active, only when insulin is present: antilipolysis increased (P less than 0.05).     

Enhancement of hatching and trophoblastic outgrowth by mouse embryos cultured in Whitten's medium containing plasmin and plasminogen

Mice were induced to superovulate and 2-cell embryos were cultured in Whitten's medium with 10 mg bovine serum albumin/ml (WM) as control, Medium WM with 2.3, 4.6, 23.1 or 46.2 micrograms plasmin/ml, Medium WM with 14.6, 29.1 or 145.7 micrograms plasminogen/ml, Medium WM with 0.1, 0.2, 1.1 or 2.2 micrograms trypsin/ml; Medium WM with 0.2, 0.3, 1.6 or 3.3 micrograms pronase/ml and Medium WM with 10% heat-treated bovine serum (HTBS). Proteolytic activities in the culture media were evaluated at the start of the culture period and 10 days later. Blastocyst formation was significantly reduced in cultures supplemented with pronase and in the two higher levels of trypsin when compared to that in Medium WM. More embryos developed to the blastocyst stage in Medium WM + 2.3 or 23.1 micrograms plasmin/ml and Medium WM + 14.6 micrograms plasminogen/ml than in Medium WM (P less than 0.05). The incidence of hatching was significantly greater in Medium WM than in all plasminogen- and plasmin-supplemented media except for Medium WM + 29.1 micrograms plasminogen/ml. Although not significantly different, hatching was lower in Medium WM and Medium WM + 0.1 microgram trypsin/ml when compared to Medium WM + HTBS. Similar numbers of embryos completed the hatching process in Media WM, WM + 0.1 or 0.2 micrograms trypsin/ml and WM + 0.3 micrograms pronase/ml. Since dissolution of the zona pellucida occurred within 96 h for embryos cultured in Media WM + 1.6 or 3.3 micrograms pronase/ml and WM + 1.1 or 2.2 micrograms trypsin/ml, hatching could not be evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro.

Efforts to improve bovine embryonic development in vitro involved study of effects of thyroid stimulating hormone (TSH) alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF), and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control), TSH (0.1, 0.5, or 1.0 micrograms/ml) or PRL (10, 100, or 1000 micrograms/ml), luteinizing hormone (LH) (0, 10, or 100 micrograms/ml) + TSH (0.1 or 0.5 micrograms/ml), and serum (20%, v/v) + 0.5 micrograms TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P less than 0.05). All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 micrograms TSH/ml (53.4%) than for other TSH treatments (P less than 0.05). The combination of TSH (0.1 and 0.5 micrograms/ml) with 10 micrograms LH/ml for IVM enabled higher proportions (P less than 0.05) of ova to fertilize (67.4 and 69.2%) than did medium alone (28.3%), LH (10 micrograms/ml) alone (54.1%) or serum + 0.5 micrograms TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 micrograms LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed in vitro to Hydergine.

Hydergine (dihydroergotoxine mesylate, Sandoz) was examined for its capability to induce chromosome damage and sister-chromatid exchanges (SCEs) in human lymphocyte in vitro. For the chromosome-aberration study, cultures set up from 6 individuals were divided into 5 groups: negative control, positive control (caffeine, 0.5 mg/ml), and Hydergine (0.1, 0.25 and 0.5 micrograms/ml). For the SCE examination, which used 8 individuals, 4 cultures were made per person in the following way: negative control, positive control (mitomycin C, 0.1 microgram/ml), and Hydergine (0.1 and 0.5 micrograms/ml). Lymphocytes were cultivated for 72 h, being exposed to the respective treatments during the final 24 h. The results showed that Hydergine induced no chromosome damage in human lymphocytes in vitro.     

The effects of plasminogen on in vitro ovine embryo development

Plasminogen activator production by ovine embryos and the effects of plasminogen on ovine embryo development and zona pellucida integrity were evaluated. Eight-cell to sixteen-cell embryos were cultured in Whitten's medium containing 0, 60, or 120 micrograms/ml plasminogen. Plasmin and plasminogen activator concentrations in the medium were determined by a caseinolytic assay. More blastocysts hatched in medium containing 60 and 120 micrograms/ml plasminogen (33 and 21%, respectively) than 0 microgram/ml plasminogen (0%; p less than 0.05). Zona pellucida dissolution time in acidified phosphate-buffered saline was less after incubation in medium with 60 and 120 micrograms/ml plasminogen (7.2 and 5.9 min, respectively) than 0 microgram/ml plasminogen (9.4 min; p less than 0.05). Plasminogen activator production was low until the morula stage, increased during morula-blastocyst transition, and remained elevated through blastocoelic expansion and hatching. Zona pellucida solubility, plasminogen activator production, and plasminogen conversion to plasmin increased as embryonic stage advanced; however, plasminogen activator production and plasmin conversion to plasmin were poorly correlated with zona pellucida solubility. The results indicate that ovine embryos produce plasminogen activator, and plasmin can increase zona pellucida solubility; however, other factors may also be involved in altering zona pellucida integrity prior to hatching.     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

Reduction of benzo(a)pyrene induced chromosomal aberrations by DL-alpha-tocopherol

The antioxidant, DL-alpha-tocopherol (vitamin E), has been demonstrated to significantly reduce the percentage of benzo(a)pyrene (BP) induced chromosomal aberrations in vitro. Chinese hamster lung (Don) and Chinese hamster ovary (CHO) cells were treated with either 1 microgram/ml or 5 micrograms/ml BP for 4 to 28 h; some cultures were treated with S9 mix activated BP. Additional cultures of Don and CHO were treated simultaneously with 100 micrograms/ml of DL-alpha-tocopherol and BP. In CHO cells 1 microgram/ml non-activated BP significantly increased the chromosomal aberration percentage above the control level. Aberrations observed included breaks, gaps, fusions, rings, dicentrics, and polyploids. Chinese hamster Don cells treated with 1 microgram/ml or 5 micrograms/ml S9 mix activated BP contained significant increases in aberration percentages above the control levels. When Don cells were treated simultaneously with activated BP and DL-alpha-tocopherol for 4 h, there was a slight decrease in the total aberration frequency to less than that of cells treated with activated BP only; however, when Don cells were treated with BP and DL-alpha-tocopherol for 28 h, there was a significant reduction in the aberration percentage below that of BP-treated cells alone. Similar results have been obtained with CHO cells treated with nonactivated BP and DL-alpha-tocopherol. The results reported here provide further evidence that antioxidants may prevent the potential mutagenic and carcinogenic effects of certain polycyclic compounds.     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic cytidine monophosphate stimulates DNA synthesis by bovine mammary tissue in vitro

Mammary gland biopsies were taken from midpregnant heifers (n = 4), cut into pieces .5 mm thick and 3 - 5 mm2 and incubated for 48 hours in Eagle's Minimum Essential Medium containing 0, .1 or 1 micrograms/ml insulin and 0, 10(-8), 10(-7), 10(-6), 10(-5), or 10(-4) M dibutyryl cyclic 3', 5', cytidine monophosphate (dbcCMP). With 0 or .1 microgram/ml insulin, dbcCMP decreased incorporation of tritiated thymidine into DNA. Similar declines in DNA synthesis were observed with sodium butyrate, suggesting that the decline was due to the butyrate rather than to a cyclic CMP-specific effect. With 1 micrograms/ml insulin, dbcCMP increased DNA synthesis. Higher levels of dbcCMP reduced DNA synthesis relative to 10(-6)M dbcCMP, as did sodium butyrate. Thus cCMP is capable of stimulating mammary growth.     

Inhibitory influence of methotrexate and vincristine on the release of cobalophilins from polymorphonuclear granulocytes

The studies have evaluated the effect of methotrexate and vincristine on the release of cobalophilins (vitamin B12 binding proteins) from resting and functionally stimulated polymorphonuclear granulocytes (PMN). Methotrexate (2.5 micrograms/ml; 5.0 micrograms/ml; 20.0 micrograms/ml; and 50.0 micrograms/ml) and vincristine (0.3 microgram/ml; 0.6 microgram/ml; 2.4 micrograms/ml; and 6.0 micrograms/ml) inhibited the cobalophilins release from resting granulocytes. This effect increased with growing concentrations of these drugs. Stimulated PMN could be shown to release cobalophilins more actively than resting granulocytes. Methotrexate (2.5 micrograms/ml; 5.0 micrograms/ml and 20.0 micrograms/ml) and vincristine (0.3 microgram/ml; 0.6 microgram/ml and 2.4 micrograms/ml) inhibited the phagocytosis-activated release of cobalophilins irrespective of the time of PMN stimulation, i.e. before or after being incubated with latex particles.     

Ciprofloxacin in the prevention and treatment of surgical infections]

A clinico-laboratory study on ciprofloxacin made by Bayer (Germany) was applied to patients with extended posttraumatic wounds and performed with the aim of preventing postoperative purulent complications in patients operated on the organs of the gastrointestinal tract. In the both groups ciprofloxacin was administered orally in doses of 500 and 1000 mg and intravenously in a dose of 200 mg. The results of the assay on ciprofloxacin sensitivity of the isolates from the wound excretion and urine showed that they were more sensitive to ciprofloxacin than to aminoglycosides and cephalosporins. 15 minutes after the intravenous administration the serum concentration of ciprofloxacin amounted to 7.5 +/- 0.9 micrograms/ml and in 6 hours it was equal to 0.45 +/- 0.45 micrograms/ml, the mean concentrations of ciprofloxacin being attained in the bile (8.7 +/- +/- 3.9 micrograms/ml), gallbladder wall (5.5 +/- 3.8 micrograms/g), liver (0.73 micrograms/g), muscles (1.93 micrograms/g) and tendon (0.15 microgram/g). After the oral administration in a dose of 500 mg ciprofloxacin was detected in the blood serum in an amount of 2.0 +/- 0.7 micrograms/ml in 1 hour and in an amount of 0.9 +/- 0.13 micrograms/ml in 6 hours. After the drug oral administration in a dose of 1000 mg the maximum concentrations were: 6.34 +/- 4.2 micrograms/ml on the average and 2.1 +/- 0.8 micrograms/ml in 6 hours (0.4 micrograms/g in the muscles, 1.4 micrograms/g in the skin and 0.34 micrograms/g in the bones). The study showed that ciprofloxacin was a highly efficient antimicrobial agent in the treatment of the complicated wound infections and the prophylaxis of the purulent complications during the postoperative period in the patients operated on gastrointestinal organs.     

A stop-flow study of intrarenal effects of atrial natriuretic factor

We performed paired series of stop-flow studies on six mongrel dogs to determine a possible nephron site of action of synthetic atrial natriuretic factor (ANF). The initial free-flow response to intrarenal infusion of 5 micrograms/min of synthetic ANF into mannitol-expanded dogs resulted in an increased urine flow rate (6.81 +/- 0.88 to 9.00 +/- 1.17 ml/min, P less than 0.05) and a 40% increase in sodium excretion (496 +/- 110 to 694 +/- 166 meq/min, P less than 0.025) when compared to paired control periods. Renal blood flow did not change, but the glomerular filtration rate increased 4% (47 +/- 5 to 49 +/- 6 ml/min, P less than 0.05). The filtered load of sodium increased 4% (P less than 0.05), and the fractional sodium excretion increased by 35% (P less than 0.01). Stop-flow experiments showed no difference in tubular sodium concentration or in the fractional sodium-to-inulin ratio at the nadir of sodium concentration, suggesting that no differences existed in distal tubular sodium handling. Further, no apparent differences were detected in collections representing the more proximal portions of the nephron. While we were able to demonstrate marked natriuresis in response to synthetic ANF, no tubular effect was discernible, and the natriuresis obtained appears to be predominantly a function of hemodynamic effects.     

心房钠泵因子对颈动脉窦压力感受器反射的易化作用

Effects of atriopeptin II (APII) on the carotid sinus baroreflex in anesthetized rats and on the sinus nerve afferent activity in the anesthetized rabbits were investigated. The results were as follows: (1) By perfusing the isolated left carotid sinus with APII (1 microgram/ml) in anesthetized rats (n = 10), the threshold pressure (TP) of the carotid baroreflex did not show any change, while the equilibrium pressure (EP), the saturation pressure (SP) and the operating range (OR) were decreased from 101 +/- 2.8 to 95 +/- 2.0 mmHg (P less than 0.05), 202 +/- 5.2 to 168 +/- 6.1 mmHg (P less than 0.001) and 128 +/- 5.5 to 93 +/- 6.3 mmHg (P less than 0.001), respectively. The function curve of the baroreflex was shifted to the left and downward with a peak slope (PS) increased during perfusing with APII. In contrast, by perfusing the carotid sinus with sodium nitroprusside (NP, 0.5 micrograms/ml), TP and EP remained unchanged, whereas SP and OR were increased from 188 +/- 6.4 to 218 +/- 6.0 mmHg (n = 6, P less than 0.01) and from 107 +/- 6.9 to 132 +/- 7.6 mmHg (P less than 0.05), respectively. The function curve of the baroreflex and its PS were not affected by NP. The sinus nerve afferent activity was quite stable with the perfusion of carotid sinus at constant intrasinus pressure (ISP) in the rabbits (n = 6) and increased during the elevation of ISP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic hormonal effects on lung maturation in fetal sheep

Cortisol has minimal effects on lung maturation in fetal sheep before 130 days gestation. To test whether there is enhancement of cortisol action by other hormones, cortisol (F), triiodothyronine (T3), epinephrine (E), prolactin (PRL), and epidermal growth factor (EGF), alone or in combination, were infused into fetal sheep for 84 h between 124 and 128 days gestation. A mixture of F + T3 + PRL, but not any combination of two hormones, increased both distensibility [1.71 +/- 0.12 (SE) ml of air/g wet wt at 40 cmH2O, V40] and stability (1.16 +/- 0.09 ml of air per g wet wt at 5 cmH2O, V5) to near full-term values, above values resulting from treatment with F alone (0.91 +/- 0.12 and 0.43 +/- 0.09 ml/g, P less than 0.01). Only F had an effect when given alone, V40 increasing (P less than 0.05). Treatment with F + T3 (0.81 +/- 0.18 ml/g) and F + E (0.77 +/- 0.07 ml/g) increased V5 above values obtained with F alone (P less than 0.05). Alveolar saturated phosphatidylcholine (SPC) was higher after treatment with F + T3 (161 +/- 52 micrograms/g), F + T3 + PRL (156 +/- 53 micrograms/g, P less than 0.05), and F + E (113 +/- 40 micrograms/g, P = 0.07) than after F (12 +/- 3 micrograms/g). We conclude that F, T3, and PRL have a synergistic effect on the development of distensibility and stability of the ovine fetal lung.     

Influences of zinc on fertilisation and development of bovine oocytes in vitro.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 micrograms added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 micrograms zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 micrograms added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 micrograms per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 microgram zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 microgram/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 microgram ml of zinc in vitro than in vivo because a concentration of 3.0 micrograms/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 micrograms/ml of zinc; however, higher concentrations were inhibitory.     

Replication patterns of the fragile X in heterozygous carriers: analysis by a BrdUrd antibody method.

The replication status of the fragile X chromosomes was studied in short-term cultures of lymphocytes from six female heterozygous carriers. The fragile X was induced by adding 0.1 microM fluorodeoxyuridine during the last 24 h of culturing. The replication status of the X chromosomes was studied using a bromodeoxyuridine (BrdUrd) antibody method. BrdUrd was added (1) at a final concentration of 0.2 micrograms/ml during the early S phase of chromosome replication (16-10 h before harvest), (2) at 0.2 microgram/ml during the late S phase (the last 6 h of culturing), (3) at 20 micrograms/ml during the early S phase, and (4) at 20 micrograms/ml during the late S phase. BrdUrd that was incorporated into replicating chromosomes was detected by using a nuclease and BrdUrd monoclonal antibody. The frequency of the fragile X was reduced by BrdUrd treatment. The degree of reduction was more severe in the 20 micrograms/ml than in the 0.2 microgram/ml series and was more severe with late S than with early S treatment. Of the early- and late-replicating fragile X chromosomes, those which were actively replicating during a BrdUrd treatment were more reduced than the others. Thus, the average rate of early and late S treatment with 0.2 microgram BrdUrd/ml was assumed to be the closest reflection of the situation in vivo. There was no correlation between the average rate of the early replicating, active fragile X and the intelligence of the heterozygous carriers studied.     

Chromosomal aberrations induced by maleic hydrazide and related compounds in Chinese hamster cells in vitro.

The cytotoxic effects and chromosomal abnormalities induced by maleic hydrazide (MH) and its salts were investigated in cultured V79 cells. MH, and its potassium (K-MH) and diethanolamine (DEA-MH) salts were tested. MH was 5--14 times more cytotoxic than its salts and almost 4.5 times less toxic than the related compound, hydrazine dihydrochloride (HDC). MH salts had very weak cytotoxicity; the LD50 values on V79 cells on exposure for 3 h in vitro were (in microgram/ml) 1100 (MH), 12 000 (DEA-MH), 20 000 (K-MH), 230 (HDC) and 10 000 (NaCl). Both MH and its salts--but neither HDC nor NaCl--caused chromosomal aberrations in cultured V79 cells. The maximal frequencies of aberrant cells in cultures exposed to the compounds for 3 h in vitro were 18% (mh at 100 microgram/ml), 18% (K-MH at 20 000 microgram/ml) and 13% (DEA-MH at 20 000 microgram/ml). Maximal frequencies observed in cultures treated with HDC or NaCl were 10% (HDC at 400 microgram/ml) and 5% NaCl at 10 000 microgram/ml). Those of positive groups were 97% (N-methyl-N'-nitro-N-nitrosoguanidine, MNNG, at 5 microgram/ml) and 16% (ethyl methanesulfonate, EMS, at 400 microgram/ml). These frequencies of MH and its salts were 3.25--4.5 times those in untreated control cells. These results suggested that MH and its salts had weak inducibili     

Functional properties of a lymphocyte subpopulation responding to low suboptimal doses of concanavalin A

Incubation of human peripheral blood lymphocytes with concanavalin A (Con A), in a low suboptimal dose (0.5 microgram/ml), results in formation of the cells that inhibit proliferation of autologous cells in cultures activated with optimal but not with suboptimal dose of the mitogen. Nevertheless, 50 micrograms/ml Con A-activated cells efficiently suppress proliferation everywhere. Cell preincubation during 18 h before Con A activation leads to a reduction of lymphocyte responses to the mitogen in cultures reactivated with 5 micrograms/ml Con A in a mixture with autologous lymphocytes, containing no mitogen. Activation of T-T helper cells providing suppressor T cells differentiation seems to take place in the presence of a low suboptimal dose of Con A. Besides, 0.5 microgram/ml Con A prevents the preincubation-induced elimination of some lymphocytes responding to an optimal dose of Con A and autologous lymphocytes.     

Antagonists of embryo-derived platelet-activating factor prevent implantation of mouse embryos

Inhibitors of platelet activation, alprazolam, iloprost and SRI 63-441, were used to demonstrate the necessity of embryo-derived platelet-activating factor (PAF) activity for the establishment of pregnancy in mice. In a splenectomized mouse bioassay 6 micrograms alprazolam inhibited, for 3 h, the thrombocytopenia induced by 0.1 micrograms PAF; 4 micrograms iloprost and 0.5 microgram SRI 63-441 were effective for 6 and 12h respectively. The administration of 2 micrograms iloprost/30 g body weight on Days 1 and 4 of pregnancy and twice daily on Days 2 and 3 caused a 50% reduction (P less than 0.0005) in the number of implantation sites in the uterus at Day 8 of pregnancy, without affecting (P greater than 0.05) the number of corpora lutea. A similar reduction in the number of implantation sites was achieved with 20 micrograms SRI 63-441/30 g body weight/day. The reduction in implantation rate was evident on Day 5 of pregnancy by visualizing the implantation sites with pontamine sky blue. SRI 63-441 had no effect on peripheral blood progesterone concentrations from Day 1 to Day 9 of pregnancy, and did not appear to inhibit implantation by blocking the preimplantation surge of oestradiol. The number and morphology of blastocysts flushed from the uterus of Day 4 inhibitor-treated mice was not different (P greater than 0.05) from the controls. The cleavage rate and morphology of embryos cultured from the 2-cell to blastocyst stage in media containing SRI 63-441 or iloprost (10 micrograms/ml) were normal, precluding a gross toxic effect. Simultaneous administration of 1 microgram PAF-acether to treated animals re-established pregnancy rates to levels not significantly different (P greater than 0.05) from the controls.