The interrelationship between mucopeptide and ribitol teichoic acid formation as shown by the effect of inhibitors

1. The biosynthesis of teichoic acid in cell suspensions of two strains of Staphylococcus aureus is partially inhibited by the same low concentrations of penicillin that inhibit mucopeptide synthesis by 90–100%. Further increase in the concentration of the antibiotic by several hundred-fold still fails to cause any greater inhibition of teichoic acid synthesis. 2. Other conditions, such as amino acid deficiency or the presence of cycloserine or 5-fluorouracil, that inhibit mucopeptide synthesis also inhibit teichoic acid formation. 3. The degree of inhibition of teichoic acid synthesis caused by relatively high concentrations (10μg./ml.) of benzylpenicillin depends critically on the age of the culture from which the cell suspensions have been prepared. 4. No significant amounts of soluble teichoic acid have been found in the fluid from cells incubated in the presence of penicillin. 5. A high proportion of the teichoic acid formed in the presence of penicillin can be removed from wall preparations at room temperature by 0·1n-ammonia. This is not true of the teichoic acid formed in the absence of penicillin. 6. The teichoic acid extracted with ammonia from preparations of cell walls made from cells treated with penicillin is excluded from Sephadex G-25, has a low molar ratio of glucosamine to phosphorus and contains muramic acid, alanine, glutamic acid, glycine and lysine. 7. The implications of these results for the mechanism of action of penicillin are discussed.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Penicillin V production by Penicillium chrysogenum in the presence of Fe3+ and in low-iron culture medium

Late-exponential-phasePenicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 μmol/L (2.2±0.6 μmol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe³⁺ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified majorP. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the β-lactam production and intracellular iron level. Neither 150 nor 300 μmol/L extracellular Fe³⁺ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H₂O₂. Nevertheless, when iron was applied in the Fe^II oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.     

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

Summary Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm² per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 μM glucosamine. A higher concentration of³⁵SO₄, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold, whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10⁻⁵ M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10⁻⁹ M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations. This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart, Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Bethesda, MD.     

Biosynthesis of peptidoglycan in Gaffkya homari. The mode of action of penicillin G and mecillinam.

The effect of the beta-lactam antibiotics penicillin G and mecillinam on the incorporation of peptidoglycan into pre-formed cell wall peptidoglycan was studied with wall membrane enzyme preparations from Gaffkya homari. Using UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) as precursors the incorporation of peptidoglycan into the pre-existing cell wall of G. homari was inhibited to an extent of 50% (ID50 value) at a concentration of 0.25 mug of penicillin G/ml. With UDP-GlcNAc and UDP-MurNAc-tetrapeptide as precursors the ID50 value was about 2500-fold greater (630 mug/ml). The inhibition by penicillin G of the incorporation of peptidoglycan from UDP-MurNAc-[14C]Lys-pentapeptide could be overcome by addition of non-radioactive UDP-MurNAc-tetrapeptide to the incubation mixture. In the presence of 5 mug of penicillin G/ml the incorporation of peptidoglycan formed from the mixture of UDP-MurNAc-Ala-DGlu-Lys-D-[14C]Ala-D[14C]Ala and non-radioactive UDP-MurNAc-tetrapeptide proceeded virtually without release of D-[14C]alanine by transpeptidase activity. The enzyme preparation also exhibited DD-carboxypeptidase activity which was only slightly more sensitive to penicillin G and mecillinam than was the incorporation of peptidoglycan into the cell wall. Since the ID50 values for the beta-lactam antibiotics are similar to the concentrations required to inhibit the growth of G. homari to an extent of 50%, the DD-carboxypeptidase must be the killing site of both penicillin G and mecillinam.     

Effects of Progesterone, β-Estradiol,and Androsterone on the Changes of Inorganic Element Content in Barley Leaves

The present study was performed to determine the changes in inorganic element content in barley leaves of mammalian sex hormones (MSH). Barley leaves were sprayed with 10⁻⁴, 10⁻⁶, 10⁻⁹, 10⁻¹², 10⁻¹⁵ M concentrations of progesterone, β-estradiol, and androsterone at 7th day after sowing. The plants were harvested at the end of 18 days after treatment with MSH solutions. The inorganic element concentrations were determined using wavelength dispersive X-ray fluorescence spectroscopy technique. Although the all MSH concentrations significantly (p < 0.05) increased the concentrations of calcium, magnesium, phosphorus, sulfur, copper, manganese, aluminum, zinc, iron, potassium, and chlorine, it decreased those of sodium concentration in barley leaves. The maximum changes in the element concentrations were obtained at 10⁻⁹ M for plant leaves treated with progesterone, 10⁻⁶ M for plant leaves treated with β-estradiol and androsterone. The present study elucidated that MSH significantly (p < 0.05) affected the inorganic element concentrations in barley leaves.     

Turnover of mucopeptide during the life cycle ofBacillus megaterium

Sporogenic and asporogenicBacillus megaterium strains, as well asBacillus cereus degraded the murein component of the cell wall labelled with¹⁴C-diaminopimelic acid to TCA-soluble compounds during growth. The rate of murein turnover was about 15% during one generation in all three cases. The addition of chloramphenicol instantaneously markedly decreased the degradation rate, whereas in the presence of penicillin the degradation proceeded at the beginning at a rate comparable with that in the control and decreased only after a certain time interval. The cell wall degradation was considerably or completely stopped during the stationary phase of growth. In sporogenic strains ofBacillus megaterium andBacillus cereus the release of mature spores was associated with a new wave of the wall degradation, during which the wall of the sporangial cell was completely digested to TCA-soluble fragments. Free spores contained practically no mucopeptide component (cortex or spore wall) originating from the wall of the vegetative cell. A possible existence of a stable fraction of the cell wall not subject to turnover was investigated by measuring the³H/¹⁴C ratio in cells labelled simultaneously with³H (or¹⁴C)-diaminopimelic acid and¹⁴C (or³H)-leucine. The ratio changed during five generations, remaining constant later. This indicates that a certain portion of murein could be stable. The murein degradation during growth was not associated with secretion or release of a significant quantity of autolytic enzymes into the medium. The wall was apparently attacked from the inside. On the other hand, the release of the spore was accompanied by an increasing autolytic activity in the medium. This latter activity reached values corresponding to 3–8 μg lysozyme/ml. The results published here were presented at the 2nd Harden Conference “Cell walls and cell membranes”, Wye, Kent, England in September 1970.     

Use of clove oil to anaesthetize freshwater amphipods

The use of clove oil as a potential anaesthetic for freshwater amphipods was examined at 20 °C. Individuals of Gammarus minus, a common species in southern Illinois, USA, spanning the entire body size range (4.3–14.3 mm), were used to test four anaesthetic concentrations varying from 1.48 × 10⁻⁴ ml ml⁻¹ to 5.9 × 10⁻⁴ ml ml⁻¹. Small-bodied individuals (mean size = 5.4 mm ± 0.27SE) were used to test additional concentrations, up to 14.7 × 10⁻⁴ ml ml⁻¹, a 10-fold span, to identify potential lethal concentrations. At the lowest concentration, time to anaesthesia and recovery was constant at all body sizes. For the three next higher concentrations, time to anaesthesia decreased with increasing concentration while recovery time increased. Activity of amphipods was not affected by the ethanol carrier. In addition, activity did not differ between amphipods that had recovered from anaesthesia and unexposed amphipods. At clove oil concentrations of 8.84 × 10⁻⁴ ml ml⁻¹ and 14.7 × 10⁻⁴ ml ml⁻¹, mortality was 7 and 40%, respectively, indicating, that 5.9 × 10⁻⁴ ml ml⁻¹ was a safe working concentration. No mortality was observed with Gammarus acherondytes, a federally endangered cave amphipod on which the protocol with 80 μl of stock was used in the field. The method enabled us to obtain information on the endangered amphipod which normally would have required the sacrifice of individuals. Thus, research can continue on species for which population numbers are low and for which basic information is needed to formulate meaningful recovery plans.     

Evidence for a novel biosynthetic pathway that regulates the ratio of syringyl to guaiacyl residues in lignin in the differentiating xylem of Magnolia kobus DC

The biosynthetic pathways to monolignols in Magnolia kobus were investigated by feeding stems with a deuterium-labeled precursor. Pentadeutero [γ,γ-²H₂, OC²H₃] coniferyl alcohol was supplied to shoots of Magnolia kobus and the incorporation of the labeled precursor into lignin was traced by gas chromatography-mass spectrometry. In addition to the direct incorporation of the labeled precursor into guaiacyl units, we detected a significant amount of pentadeuterium-labeled syringyl units with two γ-deuterium atoms. The relative level of trideuterium-labeled syringyl monomers (the result of conversion via the cinnamic acid pathway, in which two γ-deuterium atoms are removed during enzymatic re-oxidation) was negligible. Our results provide conclusive evidence for a novel alternative pathway for generation of lignin subunits at the monolignol stage and they suggest that this new pathway might be important for regulation of the composition of lignin. Received: 21 August 1998 / Accepted: 30 September 1998     

Enhancing effect of proteins derived from Xestia c-nigrum granulovirus on Mamestra brassicae nucleopolyhedrovirus infection in larvae of Autographa nigrisigna (Lepidoptera: Noctuidae) on cabbage

The insecticidal effect of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) and the enhancing activity of proteins derived from occlusion bodies (OBs) of Xestia c-nigrum granulovirus (GVPs) on the infectivity of MabrNPV were evaluated in a bioassay with second-instar larvae of Autographa nigrisigna (Walker) fed virus-applied cabbage plants. The lethal concentrations of MabrNPV achieving 50 and 95% mortality for A. nigrisigna were estimated to be 1.4 × 10⁵ and 3.1 × 10⁶ OBs/ml, respectively. When larvae were fed cabbage plants treated with MabrNPV and various concentrations of GVPs, the requisite concentration of GVPs achieving 95% mortality of A. nigrisigna was estimated to be 26.2–138.8 μg/ml in combination with 10⁴ OBs/ml MabrNPV and 8.46–24.09 μg/ml with 10⁵ OBs/ml MabrNPV. Increases in the concentration of MabrNPV or GVPs caused larval death at younger instars. A. nigrisigna has lower susceptibility to MabrNPV than M. brassicae and Helicoverpa armigera reported in Mukawa and Goto (J Econ Entomol 103:257–264, 2010). We estimated that the requisite concentration of MabrNPV for the control of A. nigrisigna was 10⁵ OBs/ml, which is a tenfold higher concentration than that for M. brassicae and H. armigera, with the increase achieved by adding 10 μg/ml GVPs.     

Concentration-dependent differntial effects of cortisol on synthesis of α-lactalbumin and of casein in cultured mouse mammary gland explants: Importance of prolactin concentration

Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka, T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml prolactin, 2.8×10⁻⁹ M to 2.8×10⁻⁷ M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase in α-lactalbumin accumulation attained in the presence of 1.4×10⁻⁸ M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin. Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin. The maximal increases were obtained in the presence of 2.8×10⁻⁶ M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration, of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be different from that involved in casein synthesis.     

Microemulsions Containing Medium-Chain Glycerides as Transdermal Delivery Systems for Hydrophilic and Hydrophobic Drugs

We evaluated the ability of microemulsions containing medium-chain glycerides as penetration enhancers to increase the transdermal delivery of lipophilic (progesterone) and hydrophilic (adenosine) model drugs as well as the effects of an increase in surfactant blend concentration on drug transdermal delivery. Microemulsions composed of polysorbate 80, medium-chain glycerides, and propylene glycol (1:1:1, w/w/w) as surfactant blend, myvacet oil as the oily phase, and water were developed. Two microemulsions containing different concentrations of surfactant blend but similar water/oil ratios were chosen; ME-lo contained a smaller concentration of surfactant than ME-hi (47:20:33 and 63:14:23 surfactant/oil/water, w/w/w). Although in vitro progesterone and adenosine release from ME-lo and ME-hi was similar, their transdermal delivery was differently affected. ME-lo significantly increased the flux of progesterone and adenosine delivered across porcine ear skin (4-fold or higher, p < 0.05) compared to progesterone solution in oil (0.05 ± 0.01 μg/cm²/h) or adenosine in water (no drug was detected in the receptor phase). The transdermal flux of adenosine, but not of progesterone, was further increased (2-fold) by ME-hi, suggesting that increases in surfactant concentration represent an interesting strategy to enhance transdermal delivery of hydrophilic, but not of lipophilic, compounds. The relative safety of the microemulsions was assessed in cultured fibroblasts. The cytotoxicity of ME-lo and ME-hi was significantly smaller than sodium lauryl sulfate (considered moderate-to-severe irritant) at same concentrations (up to 50 μg/mL), but similar to propylene glycol (regarded as safe), suggesting the safety of these formulations.     

Isolation of fatty acids from the mycelium ofPenicillium stipitatum thom and their effect on ehrlich ascites carcinoma cells

A total of 5 fractions were isolated from the mycelium ofPenicillium stipitatum Thom obtained by submerged cultivation. Three of them inhibited the incorporation of¹⁴C-labelled precursors into Ehrlich ascitic cells (EAC) at concentrations lower than 100 μg/ml. The fraction M-72-3, inhibiting mainly adenine incorporation, was further separated into 6 fractions. The highest effect on EAC cells was exerted by subfraction IV which consisted of free fatty acids; its main effective components were oleic linoleic and linolenic acid. Their cyto-inhibitory effect on EAC cells was confirmed by their application in a pure form.     

Effects of Cycloheximide upon Formation of Ribonucleic Acid Cytidylic and Uridylic Acids

Concentrations of cycloheximide as low as 3 μg/ml inhibited incorporation of labeled orotic acid or uridine into RNA cytidylic acid of soybean (Glycine max) hypocotyl sections. Even lower concentrations of this well known protein synthesis inhibitor interfered with conversion of labeled cytidine into RNA uridylic acid. Both cycloheximide and puromycin inhibited absorption of ³H-phenylalanine and its incorporation into protein, but puromycin did not significantly affect the labeling patterns of RNA cytidylic and uridylic acids when orotic acid-6-¹⁴C was fed. Results give further support to the hypothesis that cycloheximide inhibits the interconversion of uridine and cytidine nucleotides, presumably by acting as a glutamine antagonist in the glutamine-dependent reaction catalyzed by cytidine triphosphate synthetase.     

Stimulation of generative development in partly vernalized winter wheat by animal sex hormones

The influence of selected animal steroid sex hormones, on generative development of winter wheat var. Grana was investigated. Wheat plants of this variety necessitate 63-day long vernalization. Mature, isolated embryos of wheat were cultured in vitro on media containing androsterone, androstenedione, estriol, estrone, 17β-estradiol and progesterone in concentrations 10⁻⁵ and 10⁻⁶ M. They were not vernalized or vernalized for 7, 14, 21 and 28 days (5 °C, 8 h photoperiod). Investigated steroids stimulated the generative development of winter wheat by an increasing in the percentage of heading plants and accelerating the heading. The strongest effect was observed when plants were treated with steroids during the suboptimal, 21 and 28 day, vernalization. After 28 days of vernalization, 100 % heading were observed in plants obtained on the media containing androsterone and androstenedione in concentrations 10⁻⁵ and 10⁻⁶ M or estrone, 17β-estradiol and progesterone in concentration 10⁻⁵ M. Control plants showed only 8 % heading. An erratum to this article is available at .     

Neither an enhancement of autolytic wall degradation nor an inhibition of the incorporation of cell wall material are pre-requisites for penicillin-induced bacteriolysis in staphylococci

In contrast to what has been postulated, penicillin G at its optimal lytic concentration of 0.1 g per ml did not lead to a detectable activation of autolytic wall processes in staphylococci in terms of the release of uniformly labelled wall fragments from cells pretreated with the drug for 1 h. Rather a considerable inhibition of this release was observed. A similarly profound inhibition of the release of peptidoglycan fragments occurred when staphylococci pretreated for 1 h with 0.1 g penicillin per ml acted as a source of crude autolysins on peptidoglycan isolated from labelled normal cells of the same strain. This clearly demonstrated that the overall inhibition of autolytic wall processes caused by penicillin was mainly due to a decreased total autolysin action rather than to an altered wall structure. Furthermore, no substantial penicillin-induced inhibition of the incorporation of ¹⁴C-N-acetylglucosamine into the staphylococcal wall could be observed before bacteriolysis started, i. e., approximately during the first 80 min of penicillin action. These results are not consistent with any of the models hitherto proposed for the action of penicillin.Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

Inhibition of bacterial cell wall mucopeptide synthesis: A new function of RNA bacteriophage Qβ

Studies of the morphology of Escherichia coli showed that these bacilli when infected with RNA phage Qβ in broth containing hypertonic sucrose and Mg²⁺ formed osmotically labile spherical cells or spheroplasts. Phage-induced spheroplasts readily released their burst of phage when diluted into ordinary culture broth without sucrose. Investigation of the mechanism of host cell lysis revealed that incorporation of [³H]diaminopimelic acid (DAP) into the mucopeptide layer of the cell wall was markedly inhibited starting at about the midpoint of the phage replication cycle. The major site of inhibition is the DAP-containing mucopeptide layer since the synthesis of the lipoprotein-lipopolysaccharide layer, making up the bulk of the cell wall of E. coli, was not affected. A model for Qβ-mediated cell lysis is discussed which is analogous to penicillin-induced cell rupture.     

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.