In Vitro Activity of Lincomycin and Spectinomycin Against Serotypes of Avian Mycoplasma

Twenty strains of avian mycoplasma, representing 12 serotypes, were tested in vitro for their susceptibility to the action of lincomycin and spectinomycin alone and in combination. They varied in their sensitivity pattern. The ranges of minimal inhibitory concentration were 1 to 20 mug/ml for lincomycin or spectinomycin alone and 0.5/1 to 3/6 mug/ml for the lincomycin and spectinomycin combination. The ranges of minimal lethal concentration were greater with either single antibiotic than with the antibiotic combination. The amount of each antibiotic required to achieve mycoplasmacidal action of the relatively resistant strains was less with the antibiotic combination than with the single antibiotics.     

X-ray-induced mutants resistant to 8-azaguanine. II. Cell cycle dose response.

Synchronous Chinese hamster ovary cells were irradiated in G1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G1 and S phase were practically identical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 X 10(-7) (75-100 rad) to 8 X 10(-7) (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 X 10(-7) per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency (PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant (PE greater than 0.1) to 7.5 mug/ml AG, 85% to 5.0 mug/ml, and 91% to 3.5 mug/ml. Only 42% showed possible hypoxanthine-phosphoribosyltransferase (hprtase) deficiency as evidenced by HAT sensitivity (PE less than 0.1). Wild type controls exhibited PE's in 3.5 mug/ml AG of less than 0.001 and in HAT of greater than 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild type cells (D0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occurring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10-60 rad for chronic irradiation, similar to that often reported for in vivo studies.     

Sucrose-dependent spectinomycin-resistant mutants of Escherichia coli.

Spectinomycin-resistant (Spcr) mutants of Escherichia coli were isolated from nutrient agar plates containing 20% sucrose and 100 mug of spectinomycin per ml. About one-third of the Spcr mutants thus obtained were sucrose dependent (Sucd) and were classified into two types: I, those unable to grow on sucrose-free medium in the presence of spectinomycin; and II, those unable to grow on sucrose-free medium irrespective of the presence of spectinomycin. Most of these mutants were hypersensitive to antibiotics, dyes, and detergents and were abnormal in cell morphology, suggesting changes in cell envelopes. Reversion experiments indicated that the sucrose-dependent spectinomycin resistance and hypersensitivity to various chemicals were not independently induced properties. The Sucd-Spcr mutations of type I mutants were transducible by phage P1 and were mapped at the strA-aroE region.     

Effect of Selected Herbicides on Bacterial Growth Rates

Specific growth rate constants were used to evaluate the effects of selected herbicides on Erwinia carotovora, Pseudomonas fluorescens, and Bacillus sp. Comparison of growth rate constants permitted the identification of either stimulatory or inhibitory effects of these substances. E. carotovora was inhibited by 6,7-dihydrodipyrido(1,2-a:2'-c)pyrazinediium (diquat) and 4-hydroxy-3,5-diiodobenzonitrile (ioxynil) at 25 mug/ml; 1,1'-dimethyl-4,4'-bipyridinium (paraquat) at 50 mug/ml; and pentachlorophenol (PCP) at 10 mug/ml. P. fluorescens was inhibited by paraquat and PCP at 25 mug/ml and by 4-amino-3,5,6-trichloropicolinic acid (picloram) at 50 mug/ml. Stimulation of P. fluorescens was observed with 4-(methylsulfonyl)-2,6-dinitro-N,N-dipropylaniline (nitralin) at 25 mug/ml. The Bacillus species was inhibited by diquat (25 mug/ml), ioxynil (10 mug/ml), and paraquat and PCP (5 mug/ml). No significant effect of 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin), or 1,1-dimethyl-3-(alpha,alpha,alpha-trifluoro-m-tolyl)urea (fluometuron) on growth rates of the bacteria was observed at 25 and 50 mug/ml.     

A new mutation in 16S rRNA of Escherichia coli conferring spectinomycin resistance.

We report a novel mutation, C1066U in 16S rRNA which was selected for resistance to spectinomycin, an antibiotic which inhibits ribosomal translocation. The minimal inhibitory concentration (MIC) of spectinomycin determined for this mutant (15 micrograms/ml) is greater than with the wild-type plasmid (5 micrograms/ml) but lower than with the well known C1192U mutation (> 80 micrograms/ml). The C1066U mutation also increases the cells sensitivity to fusidic acid, another antibiotic which inhibits translation at the translocation stage, whereas C1192U is unchanged relative to the wild type. We discuss why the acquisition of resistance to one of these drugs is often associated with hypersensitivity to the other.     

Escherichia coli K-12 Mutants Resistant to Nalidixic Acid: Genetic Mapping and Dominance Studies

Escherichia coli K-12 strains tested so far (approximately 20) can be separated into three groups on the basis of their abilities to form colonies on nutrient agar supplemented with nalidixic acid (NAL): (i) Nal(s) or wild type (no growth at 1 to 2 mug/ml); (ii) NalA(r) (growth at 40 mug/ml or higher); and (iii) NalB(r) (growth at 4 mug/ml, but no growth at 10 mug/ml). The NalA(r) group has a spectrum of sensitivity ranging from 60 to over 100 mug/ml. All Hfr strains of the NalA(r) and NalB(r) groups transfer NAL resistance to recipient cells at genetic loci which are at 42.5 +/- 0.5 and 51 +/- 1 min, respectively, on the Taylor-Trotter map. Some members of the NalA(r) group also have the genetic locus for NalB(r). The nalA(s) allele is completely dominant to nalA(r) in a partial diploid configuration. In haploids, nalA(r)-nalB(r) is phenotypically NalA(r); nalA(r)-nalB(s) is NalA(r); and nalA(s)-nalB(r) is NalB(r). The map location of nalA and the easy differentiation between NalA(r) and NalA(s) allow this marker to be used as a counterselector in bacterial conjugation experiments.     

Bioassay for Hamycin and Amphotericin B in Serum and Other Biological Fluids

A bioassay suitable for measuring concentrations of the polyene antifungal agents hamycin and amphotericin B in biological fluids is described. By using Paecilomyces varioti as the indicator organism, sensitivity of the bioassay was found to be in the range of 0.01 to 0.02 mug/ml. A linear dose-response curve was obtained with amphotericin B; the curve for hamycin was curvilinear. In a series of assays, hamycin serum levels in the range of 0.01 to 3.5 mug/ml were measured; with amphotericin B, serum levels in the range of 0.015 to 0.175 mug/ml were measured in patients receiving orthodox intravenous medication and as high as 9.0 mug/ml in one patient treated with extraordinarily high doses of the drug.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig.

Infusion of norephinephrine (NE) (1 - 3 mug/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of prostaglandin E-like substance (PGE) at a concentration of 2.81 +/- 0.65 ng/ml in terms of PGE2. Indomethacin (3 mug/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 mug/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 - 30 mug/ml) and dexamethasone (2 - 5 mug/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 mug/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 +/- 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 mug/ml) or by hydrocortisone (100 mug/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 +/- 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 mug/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Induction of Cycloheximide-Resistant Mutants in Saccharomyces cerevisiae with N-Methyl-N′-Nitro-N-Nitrosoguanidine and ICR-170

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cycloheximide-resistant mutations in Saccharomyces cerevisiae, but few, if any, resistant mutants are induced by the acridine mustard ICR-170. Cycloheximide sensitivity in yeast is associated with the ribosome, and treatment with the antibiotic at concentrations of 2 mug/ml results in complete inhibition of protein synthesis. Missense mutations induced by MNNG probably lead to the loss of cycloheximide binding sites on the ribosome, resulting in resistance to the antibiotic without altering the activity of the organelle in protein synthesis. ICR-170, however, induced primarily frameshift mutations that would alter ribosome structural integrity, resulting in cell death rather than resistance. ICR-170 and MNNG are both mutagenic in a system in which base-pair substitution and frameshift mutations can be detected. These results indicate that cycloheximide resistance in S. cerevisiae, like streptomycin and spectinomycin resistance in Escherichia coli, can be induced by base-pair substitution mutagens but not by frameshift mutagens such as ICR-170.     

The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma.

A method is described which measures the four main prostaglandins of human semen (PGE1, E2, 19-hydroxy PGE1, and 19-hydroxy PGE2). For routine measurements E1 and E2 are measured together as are 19-OH E1 and 19-OH E2. These are measured by forming oximes in aqueous solution extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 mug/ml of 19-hydroxy Es and 30-200 mug/ml of Es.     

Platelet-activating factor alters the dynamics of prostaglandin and protein synthesis by endometrial explants from pregnant and cyclic cows at day 17 following estrus

Factors produced by bovine conceptuses alter prostaglandin (PG) and protein secretion by endometrial explants from cyclic cows and induce an intracellular inhibitor of PG synthesis. Endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows at Day 17 following estrus were incubated for 24 h with 0, 0.1, 0.5, 1 and 5 mug platelet-activating factor (PAF)/ml. Cotyledonary microsomes from parturient cows were utilized to determine levels of an intracellular/cytosolic inhibitor of PG synthesis. Endometrial explants from additional cyclic cows (n = 4) were incubated for 24 h with 0 or 5 mug PAF/ml with and without 50 muCi [(3)H]leucine. Endometrial explants (cyclic cows, n = 3) were also incubated for 12 h with each of the following treatments: 1) Control; 2) PAF (1 mug/ml); 3) lyso-PAF (2 to 10 mug/ml); 4) PAF-receptor antagonist (2 to 10 mug/ml); 5) PAF (1 mug/ml) + antagonist (2 to 10 mug/ml); 6) bovine conceptus secretory proteins (bCSP; 25 mug/ml); and 7) bCSP (25 mug/ml) + antagonist (5 mug/ml). Platelet-activating factor had distinct negative and positive dose effects on PGF and PGE-2 secretion, respectively, by explants from cyclic cows, whereas PG secretion was not altered by PAF in the endometrium of pregnant cows. Platelet-activating factor did not alter the level of an intracellular inhibitor of prostaglandin synthesis, whereas, bCSP increased the level of this inhibitor. Platelet-activating factor decreased the incorporation of [(3)H]leucine into tissue and secreted proteins for explants from cyclic cows. Lyso-PAF did not alter endometrial prostaglandin secretion. The effects of PAF but not of bCSP were blocked by the PAF-receptor antagonist. Platelet-activating factor altered PG and protein secretion by the endometrium from cyclic cows, and it may be a potential regulatory factor during early pregnancy if secreted by the bovine conceptus.     

Biological characteristics of spectinomycin-resistant strains of the tularemia pathogen

Sensitivity of 2 subspecies of the tularemia causative agent to spectinomycin, an aminoglycoside antibiotic, was studied in vitro. The MIC of the antibiotic with respect to strains 503/847 and Schu was 40 micrograms/ml and with respect to strain A-Cole 20 micrograms/ml. The frequency of spontaneous spectinomycin resistant mutants was low. The mutants grown on a medium containing spectinomycin in a concentration of 100 micrograms/ml were highly resistant to the antibiotic (at least 10000 micrograms/ml). By the main biological properties and virulence the spectinomycin resistant mutants did not differ from the initial strains.     

Surface behaviour of oestradiol-17beta.

The absorption of [3H]oestradiol-17beta from its aqueous solutions has been measured in the range 0-10mug/ml. It is found that the first adsorbed molecules are parallel to the interface and occupy 100 A2. Those adsorbed in the range 6-10 mug/ml occupy 21 A2. They are presumably associated. When the adsorption occurs in the presence of a synthetic lecithin monolayer, the molecular area is equal to 16 A2. Surface tension measurements of the solutions of oestradiol-17beta and a parallel study of their fluorescence have been performed. No association of the hormone molecules has been observed in bulk. It is concluded that surfaces and liquid monolayers may favour molecular association of the oestradiol-17beta.     

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.     

Selective stimulation by tri-iodothyronine of myocardial deoxyribonucleic acid polymerase-α in neonatal rats

Three forms of DNA polymerase (alpha, beta and gamma) were separated from isolated rat myocardial cells on the basis of template, pH and ionic requirements, sensitivity to N-ethylmaleimide and position on sucrose gradients. Tri-iodothyronine administration (20mug/100g intraperitoneally) to 3-week-old rats resulted in selective stimulation of DNA polymerase-alpha (198+/-7.1 versus 102+/-5.8pmol of [(3)H]dTMP/30min per mg of protein in untreated controls, P<0.01), with no change in polymerases-beta and -gamma. [(3)H]Thymidine incorporation into myocardial DNA was also enhanced in tri-iodothyronine-treated neonatal rats (132+/-11.2 versus 53+/-4.1c.p.m./mug of DNA in controls, P<0.001). Increased incorporation was associated with an expansion of deoxyribonucleoside 5'-triphosphate pools, especially that of dTTP (24+/-1.6 versus 10+/-1.1pmol/mg of DNA, P<0.01). Neither DNA polymerase activities nor [(3)H]thymidine incorporation were changed in 6-month-old rats in response to tri-iodothyronine. Unstimulated adult myocardial cells had DNA polymerase activities comparable with those in 3-week-old animals, but significantly lower [(3)H]-thymidine incorporation and deoxyribonucleoside triphosphate concentrations. Enhancement of both DNA polymerase-alpha activity and [(3)H]thymidine incorporation in tri-iodothyronine-treated young rats was prevented by concomitant administration of either vinblastine (1mug/g) or daunomycin (2mug/g); actinomycin D (0.1mug/g) or cycloheximide (8mug/g), on the other hand, prevented the increase in [(3)H]thymidine incorporation, but not DNA polymerase-alpha activation. These results demonstrate an age-dependent stimulation of myocardial DNA replication by tri-iodothyronine and suggest an inter-relationship between DNA synthesis and subsequent entry into mitosis.     

In vitro activity of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> antifungal protein (PAF) and its combination with fluconazole against different dermatophytes

Strains of five dermatophyte species (Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton tonsurans) were selected for testing against Penicillium chrysogenum antifungal protein (PAF) and its combination with fluconazole (FCZ). Inhibition of microconidia germination and growth was detected with MICs of PAF ranging from 1.56 to 200 mug ml(-1) when it was used alone, or at constant concentration (100 mug ml(-1)) in combination with FCZ at from 0.25 to 32 mug ml(-1). The MICs for FCZ were found to be between 0.25 and 128 mug ml(-1). PAF caused a fungicidal effect at 200 mug ml(-1) and reduced growth at between 50 and 200 mug ml(-1). Total growth inhibition with fungistatic activity was detected at 64 mug ml(-1) of FCZ for M. gypseum, T. mentagrophytes, and T. tonsurans, and at 32 mug ml(-1) FCZ for M. canis and T. rubrum. PAF and FCZ acted synergistically and/or additively on all of the tested fungi except M. gypseum, where no interactions were detected.     

Influence of glucagon, 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and triamcinolone on the arginine synthetase system in perinatal rat liver

1. The administration of triamcinolone (19-190mug/animal) to postnatal rats increased the arginine synthetase system activity 1.2-2.5-fold above control values 24h after exposure to the hormone. Cortisol (hydrocortisone), however, increased the arginine synthetase system activity only when larger (190mug/animal) or repeated daily doses were given. Glucagon (100mug/animal) stimulated arginine synthetase system activity only after the second postnatal day. None of these agents increased the activity in 19.5-21.5-day foetuses after intrauterine administration. 2. The viability of foetal rat liver explants maintained in organ culture for up to 54h was validated both by ultramicroscopic examination and by incorporation of radioactive leucine and orotic acid. 3. In organ cultures of foetal rat liver explants (18.5 days to term), triamcinolone (20mug/ml of medium) evoked a 2.8-4.3-fold increase after 24h of incubation. This increase was completely inhibited by actinomycin D (25mug/ml) or cycloheximide (10mug/ml). Cortisol (5-50mug/ml) or glucagon (0.067-67mug/ml) also increased the arginine synthetase system activity above the respective control values, but there was no increase in activity with insulin (0.05-0.25i.u./ml). 4. Maximum concentrations of glucagon (67mug/ml), dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.1mm) and triamcinolone (20mug/ml) incubated for 24h with foetal rat liver explants each produced between a two-and three-fold increase in the activity of the arginine synthetase system. Combinations of maximum amounts of glucagon and the cyclic nucleotide did not produce a greater effect than either agent alone. However, the combination of dibutyryl cyclic AMP with triamcinolone appeared to produce somewhat less than additive effects. 5. The effects of the cyclic nucleotide and triamcinolone were evident after 12h of incubation and increased steadily throughout the 24h of observation. This time-course of increased enzyme activity is very much slower than that reported for the induction of other enzymes in explant cultures of foetal rat liver.     

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.     

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.