Evaluation of Griseofulvin Binary and Ternary Solid Dispersions with HPMCAS

The stability and dissolution properties of griseofulvin binary and ternary solid dispersions were evaluated. Solid dispersions of griseofulvin and hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared using the spray drying method. A third polymer, poly[N-(2-hydroxypropyl)methacrylate] (PHPMA), was incorporated to investigate its effect on the interaction of griseofulvin with HPMCAS. In this case, HPMCAS can form H bonds with griseofulvin directly; the addition of PHPMA to the solid dispersion may enhance the stability of the amorphous griseofulvin due to greater interaction with griseofulvin. The X-ray powder diffraction results showed that griseofulvin (binary and ternary solid dispersions) remained amorphous for more than 19 months stored at 85% RH compared with the spray-dried griseofulvin which crystallized totally within 24 h at ambient conditions. The Fourier transform infrared scan showed that griseofulvin carbonyl group formed hydrogen bonds with the hydroxyl group in the HPMCAS, which could explain the extended stability of the drug. Further broadening in the peak could be seen when PHPMA was added to the solid dispersion, which indicates stronger interaction. The glass transition temperatures increased in the ternary solid dispersions regardless of HPMCAS grade. The dissolution rate of the drug in the solid dispersion (both binary and ternary) has significantly increased when compared with the dissolution profile of the spray-dried griseofulvin. These results reveal significant stability of the amorphous form due to the hydrogen bond formation with the polymer. The addition of the third polymer improved the stability but had a minor impact on dissolution.     

Inhibition of thymidine phosphorylase in vivo provides a rapid method for switching DNA labeling

Summary The interaction between nuclear ploidy and chloroplast DNA content was investigated in the unicellular green alga Chlamydomonas reinhardtii. DNA was extracted from both exponentially growing and synchronized haploid and diploid strains and analysed by CsCl equilibrium density centrifugation in an analytical ultracentrifuge. It was found that the doubling of the nuclear genome in diploids was linked to a doubling of the chloroplast DNA content per cell.     

Cellular content of ribonucleic acid and protein in Saccharomyces cerevisiae as a function of exponential growth rate: calculation of the apparent peptide chain elongation rate.

The average cellular content of ribonucleic acid and protein was determined in cultures of Saccharomyces cerevisiae growing exponentially at different rates in a variety of media. Estimations of the proportion of total cellular ribonucleic acid that is made up of ribosomal ribonucleic acid were used to calculate the average number of ribosomes per cell at the different growth rates. The fraction of ribosomes actively engaged in translation was estimated by sucrose gradient centrifugation of ribosomes and polysomes. These data were used in a calculation of the apparent time taken for the addition of an amino acid to the growing polypeptide chain; this value was found to vary linearly with growth rate over a fivefold range of doubling times.     

Griseofulvin in the treatment of dermatomycoses and onychomycoses produced by dermatophytes of the Trichophyton group

Summary Based on observations on the therapy of 128 patients affected with tinea unguium, tinea manus et pedis, tinea granulomatosa nodularis (Granuloma Majocchi), tinea cruris, tinea corporis, tinea barbae and tinea capitis due to infection with dermatophytes of theTrichophyton group, determinations were made for the absolute and relative indication of griseofulvin in the treatment of these mycoses. For each affection, comparisons were made between the therapeutic results obtained by combined therapy with oral griseofulvin (uniform daily dose 1 g for each case) and local therapy with 1 % water solution organic dyes, coal tar on the one hand, and mere local therapy as described above, on the other. For treatment, griseofulvin of different production was available: British Grisovin, Likuden and Likuden M from West-Germany, and Griseofulvin produced in the German Democratic Republic. No essential differences were found in the therapeutic effect of the individual preparations, the tolerance, however, was found to be best with Likuden. On the basis of comparisons made for the results of the individual methods of treatment, griseofulvin therapy was found to be an absolute indication of the mycotic diseases as follows: tinea capitis, tinea cruris follicularis trichophytica and tinea unguium. A relative indication was found to be tinea corporis, tinea barbae, tinea cruris, and tinea manus et pedis.All patients were subjected to microscopic and culture examination. The frequency of the individual dermatophytes was as follows:Trichophyton rubrum in 56 cases,Trichophyton verrucosum in 19 cases,Trichophyton mentagrophytes in 16 cases, andTrichophyton violaceum in 1 case. Thirty six cases showed negative cultures.In conclusion, the author recommends individual selection of patients for the griseofulvin therapy.     

Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.     

Griseofulvin: A potential agent of chromosomal segregation in cultured cells

The fungicidal compound griseofulvin (GF) induces abnormalities in nuclear division in mammalian cells cultured in vitro. For these properties it has been studied as a potential agent of chromosomal segregation. A marked effect on the dynamics of chromosomal complements was observed both on diploid and heteroploid cell lines, including hybrids produced by fusion. After treatment for three days with doses ranging from 40 to 60 μg/ml, according to the cell type, a tendency to a doubling of the chromosomal set was evident. When cells were allowed to recover in normal medium in the absence of GF a scattering of the distribution of the chromosomal numbers occured. After removal of the drug a selective advantage of the double chromosomal complements was observed on prolonged cultures. The possibility of using GF to induce chromosomal segregation for linkage studies and for chromosomal assignment is discussed.     

Shoot doubling time: A quantitative parameter for characterizing shoot cultures in vitro

Shoot doubling time is proposed as a suitable parameter for characterizing in vitro propagation rates in shoot cultures. Doubling times were estimated for cultures of black currant and apple; for black currant, doubling times ranging from 14 to 115 days were obtained, depending on the concentration of benzyladenine in the culture medium. These doubling times, which were constant for each culture, were maintained for periods of up to 76 days and could apparently be sustained indefinitely. For one subculture period only, a shoot doubling time as short as 5.6 days was obtained for black currant at high cytokinin concentration (3 × 10⁻⁵ M), but this rate could not be sustained. Shoot doubling time is a convenient parameter for use in optimizing proliferation rates in shoot cultures; its use may also facilitate investigations into the mechanisms of processes underlying shoot proliferation in vitro.     

Bilinear cell growth of Escherichia coli.

Recent electron micrograph measurements of bacterial dimensions in exponentially growing cultures of Escherichia coli support a model of bilinear increase in cell surface area and volume, with a sharp doubling in growth rate at a discrete age during the cell cycle. The results also indicate coordinate regulation of increase of surface area and volume.     

High-yield growth of E. coli at different temperatures in a bench scale fermentor

E. coli wax grown exponentially at different temperatures in a bench scale fermentor. pH was maintained at 6.8 by ammonia which served also as the nitrogen source. Glucose was introduced semi-continuously at a predetermined rate which ensured a glucose concentration of 25–50 g/liter during growth. The culture was sparged with pure oxygen. Yield constants for glucose, nitrogen, phosphorus, and oxygen were determined at the different temperatures of propagation. When all growth conditions, except temperature, were kept constant, the maximal possible yield of exponentially grown cell mass was found to be directly proportional to the doubling time. Concentrations of up to 55 g dry cells/liter culture were achieved.     

Effect of griseofulvin on lipid composition and membrane integrity inMicrosporum gypseum

The effect of griseofulvin on lipid constituents and membrane permeability ofMicrosporum gypseum has been investigated. Mycelia grown in medium containing griseofulvin (IC₅₀ concentration) possessed a lower content of total lipids, phospholipids and sterols. This inhibitory effect was further supported by decreased incorporation of [¹⁴C] acetate in total lipids, total phospholipids and sterols. Decrease in total phospholipids was also reflected to a varying extent in all individual phospholipids. An increase in the unsaturated to saturated fatty acid ratio was observed in mycelia grown in medium containing griseofulvin. Membrane permeability was affected by griseofulvin as shown by increased K⁺-efflux and greater leakage of intracellular [³²P] labelled components from prelabelled cells. Our results suggest that the antifungal activity of griseofulvin is partially due to its secondary effect on lipid constituents ofMicrosporum gypseum.     

The effect of salinity and osmotic pressure of the medium on the growth,sporulation and changes in the total organic acid content of Aspergillus flavus and Penicillium chrysogenum

Production of patulin and griseofulvin by a strain of Penicillium griseofulvum is described. Mycelial dry weight, pH and production of patulin and griseofulvin were assayed in a minimal and a complete medium; patulin or griseofulvin production was assayed in apple juice.     

The metabolic stability of ribosomal protein

Summary Measurements of the metabolic stability of ribosomal proteins of exponentially growing Escherichia coli B/r showed that their rate of degradation to free amino acids is zero to within limits of the experimental accuracy of ± 0.7% per hour or ± 0.5% per doubling period.     

Bioactive secondary metabolites from <Emphasis Type="Italic">Nigrospora</Emphasis> sp. LLGLM003, an endophytic fungus of the medicinal plant <Emphasis Type="Italic">Moringa oleifera</Emphasis> Lam.

An endophytic fungus was isolated from the root of the medicinal plant Moringa oleifera Lam. Based on analyzing the rDNA sequence, the fungus was identified as Nigrospora sp. This is the first report of the isolation of endophytic Nigrospora from M. oleifera. By bioassay-guided fractionation, four antifungal secondary metabolites were isolated from liquid cultures of the fungus Nigrospora sp. LLGLM003, and their chemical structures were determined to be griseofulvin (1), dechlorogriseofulvin (2), 8-dihydroramulosin (3) and mellein (4) on the basis of spectroscopic analyses. Compound 2, 3 and 4 were isolated from Nigrospora sp. for the first time. In vitro antifungal assay showed that griseofulvin displayed clear inhibition of the growth of 8 plant pathogenic fungi. Dechlorogriseofulvin and mellein exhibited only weak antifungal activities, whereas 8-dihydroramulosin displayed no antifungal activities.     

The Translocation of Antibiotics in Higher Plants: II. THE MOVEMENT OF GRISEOFULVIN IN BROAD BEAN AND TOMATO

The uptake and translocation of griseofulvin from water cultureby broad bean and tomato has been studied; observations werealso made on its decay in broad bean. In most cases griseofulvinwas determined by bioassay. Where possible the bioassay waschecked by chemical estimations and was found to be adequate. The amount of griseofulvin taken up by the broad bean was proportionalto the volume of water transpired for any single concentrationof the treating solution and the decay was exponentially relatedto the time of exposure in the tissues. The accumulation ofgriseofulvin in the tissue had, therefore, an exponential componentbut within the limits of error there was constant relationshipbetween accumulation and transpiration over the time periodsused in the trials. The rate of accumulation in tomato was alsoconstant. The amount of griseofulvin accumulated by both beanand tomato after a definite time was a linear function of theconcentration of the treating solution. There were two processes involved in the uptake of griseofulvinby the broad bean: (a) an initial rapid entry into the rootswhich was inhibited by sodium azide and dinitrophenol at concentrationswhich did not reduce transpiration; and (b) a prolonged uptakelinearly related to transpiration which was not affected bythese concentrations of the inhibitors.     

GRAFT INCOMPATIBILITY BETWEEN PEAR AND QUINCE: THE INFLUENCE OF METABOLITES OF CYDONIA OBLONGA ON SUSPENSION CULTURES OF PYRUS COMMUNIS

The fresh weights of suspension cultures of pear (Pyrus communis) and quince (Cydonia oblonga) increased exponentially for 30 to 40 days after subculturing. Transferring pear cultures to media in which quince cultures had grown for 10 days resulted in a 70% inhibition of callus growth. Transferring quince cultures to media in which pear cultures had grown for 10 days resulted in less than a 20% inhibition of growth. Addition of the cyanogenic glycosides amygdalin and prunasin (as 50 ppm CN ^_) killed pear cultures, while growth of quince cultures was inhibited by only approximately 50%. Addition of 50 ppm CN- severely inhibited growth of both cultures. These results indicate that 1) suspension cultures of quince release factor(s) that significantly inhibit growth of pear cultures, 2) quince cultures are relatively unaffected by metabolites released by pear cultures, 3) the severe inhibition of pear growth by quince metabolites is mimicked by the addition of cyanogenic glycosides ubiquitous to vegetative portions of quince, 4) direct cellular contact is not necessary to elicit incompatibility between pear and quince, and 5) incompatibility between pear and quince need not be associated with any particular stage of graft development.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

THE PRODUCTION OF ANTIBIOTICS IN SOIL V. BREAKDOWN OF GRISEOFULVIN IN SOIL

When griseofulvin (I; R = Cl, R '= OCH₃), a chlorine-containing antibiotic produced by Penicillium nigricans , was added to fresh garden loam, after an initial lag it disappeared rapidly. When further griseofulvin was added it was inactivated from the start and at rates which increased with each successive addition, suggesting that it was degraded biologically. The numbers of one organism, a Pseudomonas sp., increased in the soil steadily after adding griseofulvin.
When a little soil was added to a solution (pH 7·0) containing inorganic salts and griseofulvin as the sole carbon source, bioassays showed that the griseofulvin disappeared within 5 days. An organism isolated from the broth was identified as the Pseudomonas sp. thought to break down griseofulvin in soil. Griseofulvin also disappeared from a broth at pH 5·0 inoculated with soil, but at this lower pH value a dematiaceous fungus was responsible for its breakdown.
The Pseudomonas sp. also degraded two derivatives of griseofulvin, dechlorogriseofulvin (I; R = H, R'= OCH₃) and the amine (I; R = Cl, R '= NH₂). Cl^– was detected in the solutions after breakdown of griseofulvin by the Pseudomonas ; the amount present agreed well with that calculated on the assumption that all the chlorine in the griseofulvin supplied was liberated as Cl^–. Spectrophotometric examination of the solutions showed no metabolites with the aromatic ring intact, and confirmed the complete breakdown of griseofulvin suggested by the liberation of Cl-.     

Prolonged cultivation of primary chick cultures using organic buffers

Summary Primary cultures of chick embryos have been carried beyond the 50th passage in cell culture media containingN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The doubling, potential, was found to decrease twice as fast for chick cells as it did for WI-38 human cells.     

Salt-induced cell lysis of Staphylococcus aureus

Cell lysis was efficiently induced in Staphylococcus aureus by the addition of 0.3 m NaCl to exponentially growing cultures at 30°C. When cells harvested at the exponential phase were incubated in buffer with NaCl, autolysis occurred. Treatment with chloramphenicol failed to induce cell lysis by NaCl. The effects of NaCl on growing cells and harvested cells were inhibited by the addition of sodium polyanethole sulfonate, subtilisin, cardiolipin, and lipoteichoic acid. These agents diminished the activity of a cell wall-lytic enzyme liberated from the cells in the presence of NaCl. Lysis induced by salt appears to be catalyzed by a similar lytic enzyme in growing and harvested cells.