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.     

Teichoic acid-containing muropeptides from Streptococcus pneumoniae as substrates for the pneumococcal autolysin.

Pneumococcal cell walls in which the normal phosphorylcholine component of the wall teichoic acids is replaced with phosphorylethanolamine cannot absorb the homologous autolytic enzyme and are completely resistant to autolytic degradation (S. Giudicelli and A. Tomasz, J. Bacteriol. 158:1188-1190, 1984). We have now isolated and characterized soluble teichoic acid-containing muropeptides from such cell walls and tested them as substrates for the pneumococcal autolytic enzyme. Both choline- and ethanolamine-containing muropeptides were hydrolyzed to the same extent by the enzyme. Furthermore, free choline concentrations that totally inhibited the digestion of pneumococcal cell walls in vivo and in vitro were without effect when the soluble substrates were used.     

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.     

Coordinate Expression of Rubisco Activase and Rubisco during Barley Leaf Cell Development

We have utilized the cellular differentiation gradient and photomorphogenic responses of the first leaf of 7-day-old barley (Hordeum vulgare L.) to examine the accumulation of mRNA and protein encoded by the ribulose-1,5-biphosphate carboxylase holoenzyme (rubisco) activase gene (rca). Previous studies have revealed a pattern of coordinate expression of rubisco subunit polypeptides during development. We compared the expression of rubisco polypeptides and mRNAs with those encoded by rca. The mRNAs encoding both rubisco activase and rubisco are expressed exclusively in leaf tissue of 7-day-old barley seedlings; mRNAs and polypeptides of rca accumulate progressively from the leaf base in a pattern that is qualitatively similar to that of rubisco subunit mRNAs and polypeptides. The parallel pattern of rca protein and mRNA accumulation indicate that a primary control of rca gene expression in this system lies at the level of mRNA production. Light-induced expression of rca in etiolated barley follows a different pattern from that of the acropetal barley leaf gradient, however. Etiolated, 7-day-old barley seedlings contain levels of rca mRNA near the limit of detection in Northern blot hybridization assays. White light induces a 50- to 100-fold accumulation of rca mRNA, which is detectable within 30 min after the onset of illumination. In contrast, steady state levels of mRNAs encoding the small rubisco subunit are affected little by light, and mRNAs encoding the large subunit accumulate about 5-fold in response to illumination. While rca mRNA levels are low in etiolated barley leaves, levels of the protein are approximately 50 to 75% of those found in fully green leaves.     

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