Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Human impact on vegetation at the Alpine tree-line ecotone during the last millennium: lessons from high temporal and palynological resolution

Three mires and a small lake in the Swiss and Austrian Alps were studied palynologically at high resolution, covering the last 1,000, 400, 50 and 1,200 years, respectively. Methodological lessons include: (1) Sub-decadal resolution in upper, little-decomposed peat layers reveals recurrent marked fluctuations in both percentages and influx of regional tree-pollen types, reflecting variations in pollen production rather than in plant-population sizes. (2) Intermittent, single-spectrum pollen maxima in samples of sub-decadal resolution indicate pollen transport in clumps. This type of pollen transport may remain unrecognized in sections with lower sampling resolution, which may then lead to inappropriate interpretation in terms of plant-population sizes. (3) The detection of short-lived phases of human impact in decomposed peat requires sampling intervals as close as 0.2 cm. (4) PAR (pollen influx) may reflect vegetation dynamics more faithfully than percentages. Reliable PAR, however, is difficult to achieve in Alpine mires due to past human impact on peat growth, even when complex depth–age modelling techniques are used. Critical comparison of PAR with percentages is therefore essential. (5) Careful consideration of spatial scales in pollen signals (local–regional and subdivisions) is essential for a realistic palaeo-ecological interpretation. Results in terms of past human impact on vegetation are summarized as follows: (1) Trends in pollen types reflecting regional human action are in general agreement with earlier findings for the western Swiss Alps, allowing for regional differences. (2) All mires in the Alps investigated here and in an earlier study experienced human impact during the last millennium. The studied small lake, lying in sub-alpine pasture, records forest dynamics at a lower elevation since a.d. 800.     

Utility of [16 alpha-125I] iodoestradiol for autoradiography for the study of cellular and regional distribution of receptors

We demonstrate the utility of [16 alpha-125I]iodoestradiol for thaw-mount autoradiography with 2 micron and 4 micron thick sections of rat and mouse uterus, pituitary, and brain after in vivo administration. Under the conditions of the experiments, short-term autoradiography with exposure times between 3 and 14 days provides optimal cellular resolution, whereas long-term autoradiography with 1-2 months of exposure may be used to obtain topographic-regional surveys of distribution.     

Odorant stimulation of secretory and neural processes in the salamander olfactory mucosa

Topical application of the odorants guaiacol (10(-3) mol/l, 1-30 min) and 2-isobutyl-3-methoxypyrazine (IBMP, 10(-5)-10(-3) mol/l, 15 min) caused time- and concentration-dependent reductions in the secretory granule content of acinar cells of the superficial Bowman's glands (sBG) and moderate to extensive vacuolation in acinar cells of sBG and deep olfactory glands (dG). Topical application of 9.8 mg/ml scopolamine 10 min before 10(-4) mol/l IBMP significantly reduced the amount of secretory granule depletion from sBG compared to that seen with IBMP alone and resulted in less extensive vacuolation in sBG and dG acinar cells. The i.p. injection of 42 mg/kg propranolol 10 min before topical application of 10(-4) mol/l IBMP had no effect on the action of IBMP. Guaiacol and IBMP also had time- and concentration-dependent effects on the secretory activity of sustentacular cells in the olfactory epithelium. The protrusion of secretory material into the mucociliary matrix that covers the epithelial surface and vacuolation within the secretory material resulted from odorant application. Scopolamine and propranolol had no effects on the action of IBMP on sustentacular cell secretory activity. When applied in the vapor phase, guaiacol elicited action potentials recorded from individual olfactory receptor neurons; the impulse frequency was concentration-dependent and showed tonic and phasic components when the duration of stimulation was varied. Low to moderate concentrations of IBMP delivered in the vapor phase evoked monophasic negative slow voltage transients recorded from the surface of the olfactory mucosa. The amplitudes of these transients increased with increasing stimulus concentrations. Higher concentrations or longer stimulus durations evoked longer-latency positive-voltage generating processes and negative afterpotentials. The properties of the electrophysiological responses to both odorants were characteristic of responses evoked by a wide variety of 'typical' odorants.     

Long-term influence of adjuvant therapy on natural killer cell activity in breast cancer

Summary Unstimulated IFN- and IL2-stimulated (NK) cell activities were investigated in patients with breast cancer who had received either local radiotherapy alone or adjuvant chemotherapeutic treatment with CMF combined with radiotherapy 12 to 18 months previously. When tested against the primarily NK-sensitive K562 cell line, patients who had received adjuvant chemotherapeutic treatment with CMF were shown to have a significantly decreased unstimulated and IFN-stimulated NK cell activity, as compared to both patients after radiotherapy only (P<0.002) and P<0.005, respectively) and healthy control persons (P<0.05). The former group of patients also had a significantly decreased IFN-stimulated NK cell activity, when tested against the primarily NK-insensitive Chang hepatoma cell line, as compared to patients after radiotherapy only (P<0.005) and healthy controls (P<0.05). Moreover, patients after radiotherapy only proved to have a significantly increased unstimulated (P<0.01) and IFN-stimulated NK cell activity (K562: P<0.05; Chang hepatoma cell line: P<0.05), as compared to healthy control individuals. In contrast, no difference in IL2-stimulated NK cell activity was detected. The investigation for the expression of CD3 and/or Leu 19 antigens as phenotypic markers of cells with non-MHC restricted cytotoxicity showed a significantly lower percentage of cells with the CD3+ phenotype in patients with breast cancer, irrespective of the chosen post-operative treatment, as compared to healthy controls (P<0.01). Finally, patients with breast cancer who had received radiotherapy only had a significant trend towards an increased percentage of CD³+/Leu 19+ PMNC, as compared to both patients after CMF treatment (P<0.05) and healthy controls (P<0.025). We conclude that patients with breast cancer vary on a long-term basis in their NK activity and in the phenotype of their PMNC depending on their post-operative adjuvant management.Abbreviations NK natural killer cells - IFN interferon - IL 2 interleukin 2 - CMF cyclophosphamide, methotrexate, fluorouracil - ER oestrogen receptor - PMNC peripheral blood mononuclear cells - MHC major histocompatibility complex - CTL cytotoxic T lymphocytes     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.     

Autolysis-resistant peptidoglycan of anomalous composition in amino-acid-starved Escherichia coli.

Nongrowing Escherichia coli deprived of an essential amino acid continued to produce peptidoglycan at a rate approximately 30% of that of growing cells. The composition of this peptidoglycan was very different from that of growing cells and resembled that of peptidoglycan left undegraded during partial autolysis of the bacteria. Synthesis of this peptidoglycan of anomalous composition began at once upon the removal of the amino acid from the medium. Fifteen minutes of amino acid deprivation was sufficient to virtually completely prevent penicillin-induced autolytic wall degradation in vivo. During this time, although the specific activities of soluble and membrane-bound hydrolytic transglycosylases and endopeptidases remained high, the peptidoglycan produced showed decreased sensitivity to degradation in vitro. After more extensive (2-h) starvation, triggering of autolysis by chaotropic agents was also blocked. Autolysis in growing cells may be selective for peptidoglycan representing the cylindrical portion of the sacculus. It is suggested that at least part of the mechanism of the well-known lysis resistance of nongrowing E. coli is related to the deposition of structurally anomalous and relatively autolysin-resistant peptidoglycan at some strategically located sites on the bacterial surface.     

Insertional inactivation of the major autolysin gene of Streptococcus pneumoniae.

The lytA gene encoding the major pneumococcal autolysin (N-acetylmuramoyl-L-alanine amidase) was inactivated by inserting the 2-kilobase MspI fragment of pE194 containing the staphylococcal ermC gene. Stable autolysis-deficient (Lyt-) mutants and their isogenic Lyt+ parents were used in experiments designed to test possible physiological functions of the amidase. No autolysis could be induced in the mutants grown at 37 degrees C by deoxycholate, by incubation in stationary phase, or by treatment with penicillin. On the other hand, the Lyt- mutants exhibited normal growth rates and yields and normal adaptive responses during shifts from one growth temperature or nutritional condition to another. There was no evidence for impeded cell separation (chain formation). Colonies of Lyt- insertional mutants produced normal hemolytic zones on blood agar; they showed normal (high) levels of competence for genetic transformation. Lyt- mutants were also able to produce type 3 and 6 capsular polysaccharides, and such strains showed the same degree of virulence in mice as did the isogenic Lyt+ parent. The physiological function(s) of the amidase remains a puzzle.