Structural details of anthrax spores during stages of transformation into vegetative cells

Moberly, Betty J. (The University of Michigan, Ann Arbor), F. Shafa, and Philipp Gerhardt. Structural details of anthrax spores during stages of transformation into vegetative cells. J. Bacteriol. 92:220-228. 1966.-Anthrax spores in stages of dormancy, activation, germination, and outgrowth into vegetative cells were examined in an electron microscope. The fine structure proved to be much like that observed in related species of Bacillus, except for a visible alteration after heat activation and clusters of vesicle-like bodies in the cytoplasm of vegetative cells.     

Assessment of airway resistance in preterm infants during incremental inspiratory flow-resistive loading

Extrathoracic airway (ETA) stability was tested by inspiratory flow-resistive loading in 10 preterm infants to determine whether ETA collapsibility was directly related to the size of the added load. A fall in intraluminal pressure was produced by applying two inspiratory flow-resistive loads of lower (L1) and higher (L2) magnitudes. An increase in intrinsic resistance was used as an index of upper airway collapsibility. Total pulmonary resistance did not change from baseline with L1 (73 +/- 26 to 71 +/- 25 cmH2O.l-1.s) but increased significantly with L2 (72 +/- 21 to 99 +/- 34 cmH2O.l-1.s, P less than 0.02) secondary to a rise in inspiratory resistance (55 +/- 21 to 109 +/- 55 cmH2O.l-1.s, P less than 0.05). Expiratory resistance did not change significantly with either load. Proximal airway pressure was more negative with L2 than with L1 in every infant (mean -4.5 +/- 0.6 vs. -3.6 +/- 0.9 cmH2O, P less than 0.05). This study shows that the ETA of preterm infants is pressure passive at high but not at low collapsing pressures, and possible explanations include limited "active" compensation by upper airway dilator muscles and an overwhelming of the "passive" defense offered by the intrinsic rigidity of the ETA to large changes in transmural pressure.     

High resolution spatio-temporal analysis of aquatic chemical signals using microelectrochemical electrodes

Detailed understanding of chemoreceptor cell transduction andfiltering depends on precise control and thus measurement ofthe chemical stimulus. In contrast to vision and hearing, accuratestimulus measurement in chemoreception has not been possibleat biologically relevant spatial and temporal scales. In thispaper we introduce a new high-speed (200 hz) electrochemicalmethod for the direct measurement of odor signals at biologicallyrelevant space scales (10-100 µm). We tested this systemin three applications: (i) temporal and spatial features ofodor plumes, (ii) stimulus calibrations in physiological recordingchambers and (iii) boundary layer diffusion measurements withinreceptor structures.     

Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate

In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled.     

Enhanced immunocytochemical staining sensitivity in formalin-fixed and paraffin-embedded material by simultaneous use of PAP and ABC

The simultaneous use of two immunoperoxidase (IP) methods; e.g. the PAP (Peroxidase-antiperoxidase) and ABC (Avidin-Biotin) to localize a single antigen enhances the sensitivity of polyclonal antibodies (FVIII/RAg and laminin) or monoclonal antibodies (MABs) (against human B-, T-cells and macrophages) in routinely formalin fixed and in paraffin embedded material. The increased sensitivity was not accompanied by loss of specificity. With antibodies against Factor VIII related antigen (FVIII/RAg) and laminin the increased staining intensity of blood vessels were demonstrated in experimental rat transplantation tumors. The similar enhancement of immunocytochemical reactions was observed with biopsies of human brain tumors, where the monoclonal antibodies against white blood cells were used. The staining results were of comparable intensity as in snapfrozen tissue or after special embedding procedure for immunocytochemical purposes acc. to Bolton and Mesnard formol sucrose gum, sucrose paraffin; FSGSP).     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

Clearance of Exogenous Dopamine in Rat Dorsal Striatum and Nucleus Accumbens: Role of Metabolism and Effects of Locally Applied Uptake Inhibitors

In vivo electrochemistry was used to investigate the mechanisms contributing to the clearance of locally applied dopamine in the dorsal striatum and nucleus accumbens of urethane-anesthetized rats. Chronoamperometric recordings were continuously made at 5 Hz using Nafion-coated carbon fiber electrodes. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible dopamine signals were detected. Substitution of L-a-methyldopamine, a substrate for the dopamine transporter but not for monoamine oxidase, for dopamine in the micropipette did not substantially alter the time course of the resulting signals. This indicates that metabolism of locally applied dopamine to 3,4-dihydroxyphenylacetic acid is not responsible for the decline in the dopamine signal. Similarly, changing the applied oxidation potential from ±0.45 to ±0.80 V, which allows for detection of 3-methoxytyramine formed from dopamine via catechol-O-methyltransferase, had little effect on signal amplitude or time course. In contrast, lesioning the dopamine terminals with 6-hydroxydopamine, or locally applying the dopamine uptake inhibitors cocaine or nomifensine before pressure ejection of dopamine, significantly increased the amplitude and time course of the dopamine signals in both regions. The effects of cocaine and nomifensine were greater in the nucleus accumbens than in the dorsal striatum. Local application of lidocaine and procaine had no effect on the dopamine signals. Initial attempts at modeling resulted in curves that were in qualitative agreement with our experimental findings. Taken together, these data indicate that (1) uptake of dopamine by the neuronal dopamine transporter, rather than metabolism or diffusion, is the major mechanism for clearing locally applied dopamine from the extracellular milieu of the dorsal striatum and nucleus accumbens, and (2) the nucleus accumbens is more sensitive to the effects of inhibitors of dopamine uptake than is the dorsal striatum.