In Vivo and In Vitro Action of Norethindrone on Staphylococci

Norethindrone has been examined in vitro for antibacterial activity against 10 microorganisms. Turbidimetric techniques were used to assay the antibacterial activity of norethindrone. The organisms tested included Staphylococcus aureus, S. epidermidis, Micrococcus conglomeratus, Listeria monocytogenes, Streptococcus faecalis, Salmonella typhosa, Shigella flexnerii, Klebsiella pneumoniae, Escherichia coli, and Proteus vulgaris. Bacteriostatic action was shown only against the gram-positive microorganisms when they were grown anaerobically in Tryptic Soy Broth containing 10 to 50 mug of norethindrone per ml. The bacteriostatic action of norethindrone was exerted primarily during the first 8 hr of incubation and it was reduced by the presence of oxygen. Mestranol at a concentration of 1 to 10 mug/ml failed to exert any significant action on S. aureus. However, incorporation of 5 mug of mestranol per ml in the culture medium enhanced the bacteriostatic action of norethindrone on staphylococci. Enhancement of the bacteriostatic action of norethindrone could not be obtained by the addition of a concentration of 5 mug/ml of testosterone, 17alpha-estradiol, and 17beta-estradiol. Progesterone and 4-pregnen-20beta-ol-3-one under similar conditions showed an additive bacteriostatic effect when they were incorporated into the culture medium containing norethindrone. In vivo studies indicated that female, adult New Zealand rabbits, injected subcutaneously with two injections of 10 to 20 mug of norethindrone, 24 hr apart, and challenged intradermally with S. aureus 4 hr after the second injection, had fewer lesions with smaller areas of swelling and erythema as compared to control, nontreated rabbits. The protective effect of norethindrone on the development of staphylococcal lesion seemed related to hormone concentration. Thus, it was demonstrated with doses of 20, 15, and 10 mug, but not with doses of 1 and 5 mug. When the lesions were excised 48 to 92 hr after infection and when viable cell counts were made, rabbits treated with norethindrone showed significantly lower staphylococcal counts than the control rabbits. During the 1st day after infection with S. aureus, leukocytic counts of the norethindrone-treated rabbits remained normal, whereas control animals showed elevated leukocytic counts.     

Antimicrobial properties of testosterone and its intermediates

Testosterone, epiandrosterone, dehydroisoandrosterone and androsterone have been examinedin vitro for antibacterial activity againstStaphylococcus aureus, S. epidermidis, Listeria monocytogenes, Streptococcus faecalis, Escherichia coli, Alcaligenes faecalis, andSalmonella paratyphy. Turbidimetric and manometric techniques were used to assay the antibacterial activity of the above androgens. Antibacterial action was shown by epiandrosterone and dehydroisoandrosterone only against the gram-positive microorganisms when they were grown in tryptic soy broth containing 10 to 20µg of hormone per ml.The oxidation of pyruvate in the presence of 20µg/ml of epiandrosterone and dehydroisoandrosterone gave lower Qo₂ than in the absence of the hormones. In the presence of 20µg per ml of culture medium and under anaerobic conditions, testosterone and androsterone also possessed antistaphylococcal activity. In vivo tests indicate that a dose ofS. aureus twice as large was required to produce skin lesions in 50% of 6-month-old male rabbits given subcutaneously a total of 30µg of testosterone over a period of 2 weeks than in control animals. Furthermore, the size of erythema surrounding the site of injection of staphylococci or staphylococcal alpha toxin was significantly smaller in the testosterone-treated rabbits than in the control. Thus, androgens may influence resistance to furunculosis.     

Effect of Inoculum Size on In Vitro Susceptibility Testing with Lincomycin

There is disagreement in the literature as to whether lincomycin is primarily a bacteriostatic or a bactericidal agent against gram-positive cocci and also regarding the levels of activity of this agent against susceptible microorganisms. These questions were examined in a study of the effect of inoculum size on the results of tube dilution susceptibility determinations with lincomycin against 49 clinical isolates of Staphylococcus aureus and 25 strains of streptococci and pneumococci. Lincomycin was both highly active and bactericidal when tested against 40 strains of S. aureus with inocula containing a maximum of 10(4) cells per ml [median minimal inhibitory concentration (MIC), 0.78 mug/ml; median minimal bactericidal concentration (MBC), 1.56 mug/ml]. With inocula of 10(5) cells per ml, lincomycin was primarily bacteriostatic (median MIC, 1.56 mug/ml; median MBC, 12.5 mug/ml). There were further decreases in inhibitory levels and significant losses of bactericidal activity when inocula containing more than 10(7) cells were tested (median MIC, 3.13 mug/ml; median MBC > 100 mug/ml). Similar measurements with streptococci and pneumococci revealed a lesser effect of inoculum size. The mean MBC value for alpha-hemolytic streptococci increased from 0.40 to 1.05 mug/ml with an increase in inocula from 10(4) to 10(6) cells per ml, but without a marked increase in MIC values. Similar results were obtained for beta-hemolytic streptococci and pneumococci.     

R5020 enhances PGE2 stimulated steroidogenesis in cultured rat granulosa cells.

Progesterone biosynthesis and metabolization to 20 alpha-hydroxyprogesterone was stimulated in granulosa cells cultured in the presence of 20 ng/ml of follicle stimulating hormone (FSH) or increasing concentrations of PGE2 (10(-9)-10(-7)M). Concurrent treatment with the synthetic progestin R5020 (10(-6) M) enhanced the FSH or PGE2 stimulated progesterone and 20 alpha-hydroxyprogesterone accumulation in culture media, as well as delta 5-3 beta-hydroxysteroid dehydrogenase activity in granulosa cell homogenates. These findings may represent another example of an autocrine control mechanism in which the steroidogenic product of the granulosa cell exerts an ultra-short loop regulation of its own production.     

Action of Diethylstilbestrol on Staphylococci: Further Observations on the Effect of Resting Cells

Diethylstilbestrol (DS) has been shown to be active against staphylococci and other gram-positive bacteria but not against gram-negative microorganisms. The present study extends these findings. Standardized suspensions of (14)C-labeled Staphylococcus aureus serotypes III and IV and Shigella flexneri were prepared and exposed to pharmacological concentrations of DS (1 to 20 mug/ml) under diverse environmental conditions; the cells were removed by membrane filtration and the presence of radioactive substances in release to the supernatant fraction was followed by standard radioisotopic techniques. Controls were exposed similarly to the hormone vehicle alone (buffer containing 2% ethyl alcohol). DS at bactericidal concentrations above 6 mug/ml caused significant leakage of cellular radioactivity of S. aureus labeled with (14)C-glucose and (14)C-glutamic acid within 1 to 4 hr after exposure to DS. Maximum leakage of radioactivity occurred under anaerobic conditions at 37 C. Absorption studies of (14)C-labeled DS indicated that the affinity of S. flexneri for DS is markedly less than that of S. aureus. This might be one reason for the resistance of gram-negative bacteria to DS.     

Changes in oestrogen biosynthesis in preovulatory rat follicles after blockage of ovulation with pentobarbitone sodium

Follicles isolated 1 and 2 days after pentobarbitone sodium injection at pro-oestrus were incubated with C-21 steroids or aromatizable C-19 steroids. Addition of testosterone or androstenedione (50 ng/ml) increased oestradiol production by ovulation-blocked follicles, while addition of progesterone or 17 alpha-hydroxyprogesterone was ineffective. LH-stimulated oestradiol production was lower in follicles isolated 1 and 2 days after pentobarbitone sodium injection, but progesterone production was elevated compared to pro-oestrous follicles. Total steroidogenesis, measured by pregnenolone production in the presence of inhibitors of pregnenolone conversion, did not differ on the 3 days. The activity of C17-20 lyase, measured in follicular homogenates, decreased between pro-oestrus and the next day. Aromatase and 17 alpha-hydroxylase activities also decreased, but the activity of these enzymes was always considerably higher than that of C17-20 lyase. It is concluded that the decrease in follicular oestradiol production after injection of pentobarbitone sodium was due primarily to a decrease in the activity of the enzyme system responsible for the conversion of 17 alpha-hydroxyprogesterone to androstenedione, thereby limiting the amount of substrate available for aromatization to oestrogen.     

Comparison of in-vitro production of progestagens by the corpora lutea of early pregnancy of the rat and hamster

Immature rats and adult hamsters were killed on Days 2, 4 or 8 of pregnancy (Day 1 = sperm positive vaginal smear). Dispersed luteal cells (5 X 10(4) cells) were incubated for 2 h in the absence or presence of graded doses of ovine LH. In the absence of LH, incubation of rat luteal cells compared to hamster cells produced about 3-6-fold as much progesterone, 26-66 times as much 20 alpha-dihydroprogesterone and about the same amounts of 17 alpha-hydroxyprogesterone. For the rat, 1 ng LH was the minimal dose which stimulated synthesis of progesterone and 17 alpha-hydroxyprogesterone by luteal cells on Days 2 and 4 whereas 10 ng LH stimulated maximal production of progesterone by Day-8 luteal cells. As pregnancy progressed from Day 2 to Day 8, there was an inverse relationship between the levels of progesterone and 20 alpha-dihydroprogesterone accumulated by rat luteal cells. For the hamster, 1 ng LH significantly stimulated accumulation of progesterone and 17 alpha-hydroxyprogesterone by Day-2 luteal cells but not by Day-4 or Day-8 cells. Hamster luteal cells on Day 4 produced the highest levels of progesterone in response to 10 or 100 ng LH, with a maximal rate of accumulation by Day-8 cells with 10 ng LH.     

Effect of dicumarol on the growth of some soil microorganisms.

The effect of dicumarol on growth of selected soil bacteria: Azotobacter chroococcum, Arthrobacter globiformis, A. citreus and Bacillus megaterium was studied. The following minimum concentrations were inhibitory in vitro: Arthrobacter citreus--20 mug/ml., Bacillus megaterium--40 mug/ml., Azotobacter chroococcum--40 mug/ml. Arthrobacter globiformis--70 mug/ml. Cells of all microorganisms studied grown in the presence of dicumarol developed aberrant morphological forms.     

Responses of Staphylococci to Androgens

The reported regulatory activities of hormones on mammalian cells suggest that a hormonal effect may be of importance in the host-parasite relationship of staphylococci. Male 6-month-old rabbits of similar genetic constitution were given subcutaneously 20 mug of androgens in saline containing 1% ethyl alcohol. Control rabbits received 1% ethyl alcohol in saline. At 5 to 10 min after administration of androgens, the rabbits were bled by cardiac puncture, and the serum was separated and incubated with standardized suspensions of Staphylococcus aureus serotypes I to XIII. It was found that S. aureus grew more luxuriantly in the sera of the control rabbits than in the sera of androgen-treated animals. With tryptic soy broth as a culture medium, a concentration 150- to 300-fold (30 to 40 mug/ml) higher than that achieved in the blood of an androgen-treated rabbit was required to yield an equivalent effect. In addition, the androgen-staphylococcal interaction has been studied with regard to experimentally induced furunculosis, the uptake of androgens by staphylococci and steroid molecular structure and antimicrobial activity. The data indicate that androgens may play a role in protection against staphylococcal infection.     

Lactic Acid Utilization by the Cutaneous Micrococcaceae

Human cutaneous staphylococci and micrococci utilized lactic acid as an energy source on a minimal medium. Propionic acid was not utilized, but l(+)-lactic acid and pyruvic acid could replace ld-lactic acid as a substrate. Selected strains of cocci were inhibited more by the l(+) and d(-) forms of lactic acid than the balanced ld form, particularly at pH 5.6. With proper dilution of substrate, lactic acid was utilized by selected strains in the presence of 10 mug of oleic and palmitic acids per ml.     

Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm

Progesterone and 17 alpha-hydroxyprogesterone (but not other steroids such as testosterone, corticosterone, beta-estradiol, estrone, dehydroepiandrosterone, 20 alpha-hydroxypregnen-3-one, androstenedione, and pregnenolone) were shown to cause an immediate increase, in free cytosolic calcium ([Ca2+]i) in both capacitated and noncapacitated human sperm, using the fluorescent indicator fura 2. Significant increases in [Ca2+]i were observed with 10 ng/ml progesterone, while maximum effects were seen with 1 microgram/ml progesterone. Two other steroids 11 beta-hydroxyprogesterone and 5 alpha-pregnane-3,20-dione exhibited significant activity to increase [Ca2+]i. This increase in [Ca2+]i elicited by progesterone was entirely due to Ca2+ influx from the extracellular medium since the increase in [Ca2+]i was blocked by the Ca2+ chelator EGTA (2.5 mM) and the Ca2+ channel antagonist La3+ (0.25 mM) when added to the medium containing 2.5 mM Ca2+. Progesterone also stimulated the uptake of Mn2+ into sperm as measured by the quenching of fura 2 fluorescence. Progesterone has been found in human follicular fluid at levels capable of stimulating increases in [Ca2+]i. The similarities in responses induced by human follicular fluid and progesterone an increase in [Ca2+]i, and hence the acrosome reaction, is progesterone and/or 17 alpha-hydroxyprogesterone. Progesterone (1 microgram/ml) did not increase [Ca2+]i in somatic cells such as adipocytes, hepatocytes, Balb/c 3T3 cells, normal rat kidney, or DDT1 MF-2 cells. The effects of these progestins to increase [Ca2+]i, by activating a receptor-operated calcium channel, is the first report of such an activity in sperm. This phenomena possibly opens up a new field of steroid action in the area of sterility, fertility, and contraception at the level of the sperm.     

In Vitro Antiviral Activity of Mycophenolic Acid and Its Reversal by Guanine-Type Compounds

With the agar diffusion test and BS-C-1 cells, mycophenolic acid was found to give a straight-line dose-response activity in inhibiting the cytopathic effects of vaccinia, herpes simplex, and measles viruses. Plaque tests have shown 100% reduction of virus plaques by mycophenolic acid over drug ranges of 10 to 50 mug/ml and virus input as high as 6,000 plaque-forming units (PFU) per flask. Back titration studies with measles virus inhibited by mycophenolic acid have indicated that extracellular virus titers were reduced by approximately 3 logs(10) and total virus was reduced by 1 log(10). The agar diffusion test system lends itself readily to drug reversal studies. Mycophenolic acid incorporated into agar at 10 mug/ml gave 100% protection to virus-infected cells. Filter paper discs impregnated with selected chemical agents at concentrations of 1,000 mug/ml (20 mug per filter paper disc) were placed on the agar surface. Reversal of the antiviral activity of mycophenolic acid was indicated by virus breakthrough in those cells in close proximity to the filter paper disc. Chemicals showing the best reversal of the antiviral activity of mycophenolic acid were guanine, guanosine, guanylic acid, deoxyguanylic acid, and 2,6-diaminopurine. The reversal of antiviral activity was confirmed by titrations of virus produced with various amounts of both mycophenolic acid and guanine present and by isotope tracer methods with uptakes of labeled uridine, guanine, leucine, and thymidine in treated and nontreated, infected and noninfected cells as parameters. All antiviral effects of mycophenolic acid at 10 mug/ml could be reversed to the range shown by untreated controls by the addition of 10 mug/ml of those chemicals exhibiting reversal activity.     

Some Active Derivatives of Penicillin

The antibacterial activities of a number of amide derivatives of penicillin against both penicillin-sensitive and penicillin-resistant cultures were determined. Several of them were found to possess significant inhibitory activity against certain gram-positive bacteria. The amides, although resistant to the destructive action of beta-lactamase, did not protect G in competitive experiments. One derivative, the O-benzylhydroxamide of penicillin G, was active against six or eight penicillin-resistant strains of Staphylococcus aureus (minimal inhibitory concentration, 0.2 mug/ml or less), but was found to have only a minimal in vivo activity against mouse Streptococcus infections.     

Mechanism of Action of the Gonadal Steroids Producing Diminution of Growth of Staphylococcus Aureus

S ummary . Studies were undertaken to characterize the mechanism of action of the gonadal steroids responsible for decreasing growth of Staphylococcus aureus in vitro. Progesterone or testosterone at 20 μg/ml significantly increased the leakage of ¹⁴C activity from staphylococci pre-loaded with ¹⁴C-glucose. This enhancement of leakage was not detected with Gram negative micro-organisms. Hormonal diminution of total uptake of alanine was relatively independent of temperature and of the phase of culture. Anaerobiosis increased the steroidal diminution of alanine uptake c. 2-fold. Fraction-ation of staphylococci following exposure to various ¹⁴C-substrates in the presence of progesterone at 40 μg/ml did not reveal any distinctive influences on macromolecular syntheses. Entry of the labels into cellular pools, however, was altered for 8 of the 10 substrates tested. Exchange experiments detailed the effects of steroids on the efflux of internal alanine and lysine. With progesterone at 40 μg/ml, alanine effluxed from the internal pool 3 times as fast as from the corresponding controls. The opposite effect occurred with lysine and progesterone depressed its exit rate. The stepwise removal of cellular constituents indicated a preferential binding of hormones to cell wall components. Using ¹⁴C-progesterone or ¹⁴C-diethylstilbestrol, 24% and 29%, respectively, of the added hormone was firmly bound to mucopeptide preparations, compared to 1–5% bound to whole cells or isolated cell walls. We suggest that the hormones interfere with the integrated functioning of membrane-associated processes.     

Semisynthetic Penicillin 6-[d(—)-α-Carboxy-3-Thienylacetamido] Penicillanic Acid Active Against Pseudomonas In Vitro

The activity of 6-[d(-)-alpha-carboxy-3-thienylacetamido] penicillanic acid, BRL2288, was determined against Pseudomonas aeruginosa and various gram-negative bacilli. The majority of Pseudomonas strains (89%) were inhibited by 100 mug of the antibiotic per ml. BRL2288 is twofold more active than carbenicillin against Pseudomonas at 100 mug/ml or less. Among Enterobacteriaceae tested, 87% Enterobacter and 87% of Proteus mirabilis strains were inhibited by 25 mug/ml or less. Indole-positive Proteus were inhibited by 10 mug/ml or less. Fifty-five per cent of ampicillin-resistant Escherichia coli were inhibited by 100 mug/ml. Klebsiella were uniformly resistant. BRL2288 is not hydrolyzed by most resistant Pseudomonas, but it is destroyed by the beta-lactamases of E. coli and P. mirabilis. The antibiotic shows synergy with gentamicin but not with penicillinase-resistant penicillins such as cloxacillin. Activity of BRL2288 against gram-positive organisms is two- to eightfold less than that of ampicillin or benzylpenicillin G.     

The non-genomic effects on Na+/H+-exchange 1 by progesterone and 20alpha-hydroxyprogesterone in human T cells

Progesterone is an endogenous immunomodulator and can suppress T-cell activation during pregnancy. We have previously shown that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)-induced T-cell proliferation. The presence of amiloride-sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20alpha-hydroxyprogesterone (20alpha-OHP). Furthermore, 20alpha-OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone-BSA demonstrated non-genomic action via plasma membrane sites. Finally, co-stimulation with PHA and progesterone or amiloride, (5-(N, N-dimethyl)-amiloride, DMA), inhibited PHA-induced T-cell proliferation, but this inhibition did not occur with 20alpha-OHP and PHA co-stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA-induced T-cell proliferation. This is the first study to show that progesterone causes a rapid non-genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20alpha-OHP. The inhibition of NHE1 leads to immunosuppressive T-cell proliferation and suggests that progesterone might exert a major rapid non-genomic suppressive effect on NHE1 activity at the maternal-fetal interface in vivo and that 20alpha-OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface.     

Heparin-releasable lipase activity of rat adrenals, ovaries and testes.

The presence of NaCl-resistant, neutral triacylglycerol hydrolase (lipase) activity in rat adrenal gland, ovary and testis was studied. Both adrenals and ovaries but not testes were found to contain such a lipase. The activity of the enzyme in the adrenal gland was lowered during cortisol treatment and hypothyroidism. An elevated adrenal lipase activity was found during hyperthyroidism. Pseudo-pregnant and lactating rats had higher ovarian lipase activities than cyclic rats. Ovarian lipase activity in lactating rats was positively correlated with the serum concentrations of progesterone and of 20 alpha-hydroxyprogesterone and negatively correlated with the high-density-lipoprotein non-esterified cholesterol concentration. The lipase activity of adrenals and of ovaries was largely releasable from these organs by heparin and could be inhibited by an antibody against heparin-releasable liver lipase. This indicated that the lipase is extracellularly located and is similar to 'liver' lipase. A possible role of this lipase in adrenals and ovaries is discussed.     

New Antibacterial Agent Isolated from the Avocado Pear

A group of eight new long-chain aliphatic compounds recently isolated from the avocado and some derivatives thereof were tested for antibacterial activity on 13 different species of bacteria and a yeast. Some of these compounds inhibited the growth of microorganisms. 1,2,4-Trihydroxy-n-hepadeca-16-en was found to be the most active, inhibiting certain gram-positive bacteria at 4 mug/ml.     

Physiological and biochemical effects of the mycotoxin patulin on Chang liver cell cultures.

Patulin exhibits both cytotoxic and cytopathic effects on cultured Chang liver cells. The LD50 found was 1.85 mug per ml of patulin. Effects on growth were observed with as little as 0.1 mug per ml of patulin; a 50% reduction in growth was observed at 0.38 mug per ml of patulin. Using a challenge dose of 2.5 mug per ml of patulin, the cytotoxic effect was reversible after an exposure of 10 min, but was not reversible after 20 min. Protein synthesis was depressed after 60 min and RNA synthesis after 20 min of contact with patulin. Neither protein nor RNA synthesis was completely inhibited after 260 min.