Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells.

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min. and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8-20 h period. Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstanital evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.

Water in normal muscle and muscle with a tumor.

The total water content, the amount of non-freezable water, and the Na-+ and K-+ contents in the gastrocnemius muscle of albino mice with and without a solid tumor were determined. The spin-lattice relaxation time (T1) for the water protons in the two kinds of muscle were measured at six resonance frequencies ranging from 4.5 to 60 MHz over the temperature range +37 to minus 65 degrees C. Quantitatively calculated T1 values are given. The difference in T1 for the two types of muscle at temperatures above minus 5 degrees C is attributed to the difference in the distribution ratio of water between hydration and free states, and bears no direct relation to the concentration of Na-+.

Novel pyrazolo, isoxazolo, and thiazolo steroidal systems and model analogs containing dimethyoxylaryl (or dihydroxylaryl) groups and derivatives. Synthesis, spectral properties, and biological activity.

The total syntheses of a series of vicinal-substituted dimethoxy and dihydroxy heterosteroids of the equilenin type and model analogs are described. A novel class of pyrazolo steroidal N-glucosides has also been synthesized. Compounds prepared were screened in vitro for growth inhibition of different microorganisms. Of these, 1-alpha-d-glucopyranosyl-4,5-dihydro-7-methyoxy-1H-benz[g]indazole tetraacetate (3) was quite active. For example, N-glucoside 13 inhibited the growth of Bacillus subtilis, Pseudomonas fluorescens, Staphylococcus aureus, and KB cells at moderate concentrations.

Synthesis and structure-activity relationships of heterocyclic compounds containing a trimethoxyarene function.

Pyrazole-, isoxazole-, and pyrazolone-containing systems were prepared from 3,4-dihydro-5,6,7-trimethoxy-1(2H)-naphthalenone, 3,4-dihydro-6,7,8-trimethoxy-1(2H)-naphthalenone, and 3,4-dihydro-6,7,8-trimethoxy-1(2H)-phenanthrone. Primarily, the pyrazoles displayed inhibition of growth in the microbial screens and in tissue culture. Correlation of the heteroatom distances between the oxygen atoms of two methoxy groups and a nitrogen atom in the pyrazole function with the percent plating efficiency on KB cell growth suggests increased inhibition as the (OA-N)/(OB-N) ratio deviates from one. No trend was observed in relating the OA-N-OB angle and activity for the examples studied.

Synthesis and antimicrobial activity of azasteroid-type compounds and related systems. Effect of hydrophilic and lipophilic groups on activity.

Pyrazole-, pyrazolone- and isoxazole-containing systems were prepared from 3,4-dihydro-6-(hexyloxy)-1(2H)-naphthalenone, 3,4-dihydro-6-(hexadecyloxy)-1(2H)-naphthalenone,3,4-dihydro-6(2-dimethylaminoethyloxy)-1-(2H)-naphthalenone, 3,4-dihydro-7-hexyloxy-1(2H)-phenanthrone, and 3,4-dihydro-7-(2-dimethylaminoethyloxy)-1(2H)-phenanthrone. A number of compounds derived from 7, 8-dihydro-5(6H)-quinolinone were also synthesized and characterized. Both hydrophilic and lipophilic groups were incorporated into certain systems as well as cidal groups. The compounds were screened for their in vitro inhibitory activity against Bacillus subtilis and Pseudomonas fluorescens. Structure-acitivity relationships among the molecular systems are discussed.

Synthesis and biological activity of some derivatives of thiochroman-4-one and tetrahydrothiapyran-4-one.

A small series of pyrazoles and isoxazoles derived from thiochroman-4-one has been synthesized and characterized. The compounds were examined for their in vitro inhibitory activity against Bacillus subtilis and Pseudomonas fluorescens. Among the tested compounds the pyrazole derivative from thiochroman-4-one was found to be the most effective inhibitor of growth of B. subtilis. Extensive H NMR analysis was recorded for all compounds.

Antibacterial activity of 15-azasteroids alone and in combination with antibiotics.

A new class of 15-azasteroid analogues has been synthesized and tested for antimicrobial activity. The compounds 1, 10, 11, 11a-tetrahydro-7-methoxy-11a-methyl-2H-naphth (1,2-g) indol (methoxyimine) and 1,10,11,11a-tetrahydro-11a-methyl-2H-naphth (1,2-g) indol-7-ol (hydroxyimine) inhibit the growth of Bacillus subtillis and Escherichia coli at concentrations as low as 10-5 M. Addition of either compound to the growth medium casued a rapid inhibition in the transport of radioactive glucose, uracil and several amino acids. The inhibition of growth and substrate transport was reversed following removl of the steroid from the medium. The evidence is consistent with a site of steroid action at the cell periphery. Combining the methoxyimine with polyor circulin at subinhibitory concentrations produced greatly enhanced antimicrobial activity against Pseudomonas fluorescens. Similar action was observed against B. subtilis when the azasteroid was combined with vancomycin or chloramphenicol. The inhibitory action of other antibiotics such as penicillin or erythromycin was not affected by addition of the test compound. The results suggest formation of a molecular complex between the azasteroid and antibiotic which is responsible for the enhanced biological activity.

15-Azasteroid blockage of cell permeability and mitochondrial respiration.

The 15-azasteroid, 1,10,11,11a-tetrahydro-11a-methyl-2H naphth (1,2-g)indol-7-o1, inhibits the growth of the cell culture lines KB and L-M as well as several strains of bacteria. The inhibition of growth is reversed following removal of the steroid from the growth medium. Using in vitro grown L-M cells, the compound inhibited the transport of amino acids and uracil. The action was non-detergent like and at least 100 times more effective in terminating metabolite transport than sodium azide. The azasteroid inhibited the oxidation of glutamate in isolated rat liver mitochondria. The oxidation of succinate was not effected by the azasteroid alone but in the presence of glutamate, the azasteroid uncoupled the oxidation of succinate from the ADP-ATP control. It is suggested that the azasteroid may be acting directly on the electron transport system and/or acting indirectly through membrane perturbations which disrupts the electron transport process.

Simple agar block slide culture suitable for varying the environment of bacteria during time-lapse cinephotomicrography.

A simple paper wick agar block slide culture is described for use in experiments which vary the chemical environment of bacteria during time-lapse cinephotomicrography.

The role of multivalent cations in the uptake and oxidation of glucose by Pseudomonas fluorescens.

The effects of Mg(2+), Mn(2+), Ca(2+) and Fe(3+) on the uptake and oxidation of glucose in induced and non-induced Pseudomonas fluorescens cells were studied. Mg(2+) is the most effective single metal ion in the uptake of glucose by the cell, and it functions in membrane stabilization and enzymic activity. Ca(2+) will substitute for Mg(2+), but Mn(2+) and Fe(3+) will not.

Effect of carbon source during growth on sensitivity of Pseudomonas fluorescens to actinomycin D.

Sensitivity to actinomycin D(AD) varies in Pseudomonas fluorescens cells grown in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity in glucose-grown cells. Succinate-grown cells bound 2-3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lipopolysaccharide masks the binding sites and, therefore, is responsible for the difference in binding of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic.

Quantitation of the action of ethylenediaminetetracetic acid and tris(hydroxymethyl)aminomethane on a gram-negative bacterium by vancomycin adsorption.

Changes in the cell wall of a species of Flavobacterium can be quantitated by measuring vancomycin adsorption. Treatment of the cells with ethylenediaminetetraacetic acid or tris(hydroxymethyl)aminomethane (or both) increased adsorption of the antibiotic.

Biological, proton-magnetic-resonance- and ultraviolet-spectroscopic evidence for a molecular complex of actinomycin D and 10,11-dihydro-3H-naphth (1,2-g)indazol-7-ol.

A proton-magnetic-resonance study of the complex formed between actinomycin D and 10,11-dihydro-3H-naphth[1,2-g]indazol-7-ol strongly suggests that the indazole lies above the phenoxazine ring of actinomycin D. The complex can be destroyed by addition of dimethyl sulphoxide or dimethylformamide. The actinomycin D-indazole complex inhibits growth of Pseudomonas fluorescens and raises the thermal denaturation response of DNA. These data support the hypothesis that a molecular complex is formed which readily inhibits cell growth and interacts with DNA.