Ventilatory and blood gas dynamics at onset and offset of exercise in the pony

The purpose of these experiments was to examine the temporal pattern of arterial carbon dioxide tension (PaCO2) to assess the relationship between alveolar ventilation (VA) and CO2 return to the lung at the onset and offset of submaximal treadmill exercise. Five healthy ponies exercised for 8 min at two work rates: 50 m/min 6% grade and 70 m/min 12% grade. PaCO2 decreased (P less than 0.05) below resting values within 1 min after commencement of exercise at both work rates and reached a nadir at 90 s. PaCO2 decreased maximally by 2.5 and 3.5 Torr at the low and moderate rate, respectively. After the nadir, PaCO2 increased across time during both work rates and reached values that were not significantly different (P greater than 0.05) from rest at minute 4 of exercise. Partial pressure of O2 in arterial blood and arterial pH reflected hyperventilation during the first 3 min of exercise. At the termination of exercise PaCO2 increased (1.5 Torr) above rest (P less than 0.05), reaching a zenith at 2-3 min of recovery. These data suggest that VA and CO2 flow to the lung are not tightly matched at the onset and offset of exercise in the pony and thus challenges the traditional concept of blood gas homeostasis during muscular exercise.     

Juvenile/total foliage ratios inEucalyptus nitens and the growth of stands and individual trees

Summary Individual trees and stands of two provenances of Eucalyptus nitens which have marked differences in retention of juvenile foliage were studied in four plantations at different elevations. The proportion of juvenile to total foliage and growth was measured at the end of the 2nd, 3rd and 4th year from establishment. Between the ages of 2 and 4 years annual stem volume increment increased in proportion to the amount of juvenile foliage retained. By age 4 years, stem volume of trees of the juvenile persistent form was significantly larger than that of the early adult form. Increasing differences in height growth with age between provenances, which were highly significant across sites by age 4, contributed to these differences in performance. There was some evidence that the largest trees of the juvenile-persistent form were those which combined mature foliage above juvenile foliage for the longest period during the transition from juvenile to mature foliage. In the early-adult form the largest trees were those which completed the transition to mature foliage rapidly. There was no difference in the ratio of foliage mass to basal area between the two forms. It is suggested that the faster growth of the juvenile-persistent form is related to higher leaf area index and not to foliage type. A provenance of E. globulus which had a higher retention of juvenile foliage at age 4 than a second provenance had a lower stem volume, thus indicating that in this species early growth rate is not determined by foliage type.     

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.      

Ultrastructural localisation of acid phosphatase,non-specific esterase and β-glucuronidase in the midgut epithelium of Tenebrio molitor,Schistocerca gregaria and Carausius morosus

Summary Acid phosphatase, non-specific esterase and -glucuronidase have been localised in the midgut epithelium of three species of insect using naphthol esters as substrates and triphenyl-p-amino-phenethyl lead as coupling salt. In all three species acid phosphatase and -glucuronidase appear to be confined to primary and secondary lysosomes. Non-specific esterase activity was demonstrated within membrane-enclosing bodies in all three species, associated with lipid droplets in T. molitor and C. morosus and with an unidentified intranuclear structure in C. morosus.     

Cytochemical studies of the types and localization of acid phosphatases in the various regions of the midgut epithelium of Carausius morosus

Synopsis Cytochemical studies on the localization and substrate specificities of acid phosphatase activities in the epithelial cells of the midgut ofCarausius morosus have revealed the presence of two distinct types of phosphatases. Acid naphthol AS-BI phosphatase activity was present at particulate (lysosomal) sites in all regions of the midgut and its activity was particularly high in the pear-shaped organs. Acid -glycerophosphatase of low activity was present in the mid and posterior midgut regions, but was absent from the pearshaped organs. In the anterior region of the midgut, acid -glycerophosphatase activity could only be found associated with the concentrically laminated vesicles.     

Effects of stimulation and rest on the ultrastructure of the excitatory neuromuscular junctions of Locusta migratoria L.

Summary The terminals of the fast axon on extensor tibiae muscle fibres of Locusta were examined in untreated nerve-muscle preparations and in preparations stimulated electrically at frequencies varying from 0.5 to 100 Hz. The ultrastructure of the terminals in preparations stimulated at the lower range of these frequencies, which induce twitch contractions of the muscles, is similar to that of the controls. Stimulation at the higher frequencies induced tetanic muscle responses and rapid fatigue of the muscles after which they would not respond again to high frequency stimulation for about 1 h. This loss and recovery of the responses of the muscles is correlated with changes in the ultrastructural appearance of the terminals, in particular in the number and shape of the synaptic vesicles. The ultrastructure of these recovering axon terminals closely resembles that of the controls.R.P. Botham gratefully acknowledges the SRC for financial assistance     

Recovery after defoliation in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> saplings: respiration and growth

Key message

Recovery after partial defoliation and/or debudding treatments was found to be more closely related to the release of latent buds rather than temporal changes in leaf-level respiration and carbon uptake.

Abstract

Despite the importance of respiration in the overall carbon balance of plants, recovery after defoliation and debudding has been largely related to changes in carbon uptake; the significance of respiration has received much less attention. Growth, biomass and leaf-level carbon balance (both photosynthesis and dark respiration at night) responses of young Eucalyptus globulus potted-saplings to debudding (B), partial defoliation (D) and combined B&D treatments were assessed over a 12-week recovery period. Light-saturated photosynthetic rates (A ₁₅₀₀) were asynchronous with night respiration rates (R _dark) throughout the course of the experiment; 5 weeks after defoliation, significant increases in A ₁₅₀₀ were accompanied by concomitant increases in R _dark in the B&D and B and D treatments. By week 8, while A ₁₅₀₀ returned to control values, R _dark had increased, particularly in the B&D treatment. Saplings in the B and D treatments showed full recovery with growth, biomass and leaf area being similar to control saplings by week 12. In contrast, saplings in the B&D treatment appeared unable to compensate for the combined removal of all buds and 35 % leaf area as evidenced by slowed height increments and reductions in total biomass of >30 %. Simple modelling of whole-plant net CO₂ uptake showed that saplings in the B&D treatment fixed 20 % less CO₂ than the other treatments at week 12, suggesting that recovery following this treatment and the D treatment was dependent on changes in total leaf area development and whole-tree assimilation rather than differences in assimilation or respiration per unit foliage area. Increased biomass allocation to bud in weeks 5 and 8 suggested that the pattern of refoliation after defoliation and debudding was related to changes in tree architecture from the release of latent buds.

     

The survival and growth of an environmental Klebsiella isolate in detergent solutions

A Klebsiella isolate (G1) was capable of survival and growth in an environment containing high levels of anionic (15.6%) and non-ionic detergent (7.8%). Cell numbers were monitored by traditional viable plate counts (culturability), and by staining with Rhodamine 123 (vital counts) and Acridine Orange (total counts). On inoculation into detergent solutions, only 10% of cells retained vitality (rhodamine 123) after 24 h. Of those, only 1% were able to form colonies on artificial culture media. Under low nutrient conditions (TOC < 0.89 mg l - 1), no recovery was observed over a 96 h period, but addition of nutrients (TOC 3.84 mg l - 1) allowed recovery within 48 h. Such cells were initially (24 h) elongated, but subsequently (144 h) returned to their normal size. In the presence of detergent without added nutrient, cell size was reduced after 144 h. After adaptation to the detergent environment (24 - 48 h), cell numbers in detergent with added nutrient broth (vital, viable and total) rose, until levels equivalent to those of a detergent-free control were attained. Detergent solutions provide a stressful environment. Nutrient levels in the detergent solutions affected cell size and the ability of the Klebsiella (G1) to survive and grow.     

Functional analyses of AmpC beta-lactamase through differential stability.

Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the beta-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for beta-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other beta-lactam binding proteins as well.     

Binding of concanavalin A to Chinese hamster ovary cells after storage in liquid nitrogen.

The binding of Concanavalin A to the surface of Chinese Hamster Ovary (CHO) cells after storage in liquid nitrogen was studied by a number of techniques. Electron microscope cytochemistry revealed that cells frozen at 1.7 °C/min resulting in high viability had a Con A positive surface coat similar to non-frozen control cells whereas those that had been frozen at 200 °C/min, resulting in low viability, exhibited a range of staining patterns with many cells showing a loss of the coat. However, the binding of I¹²⁵-labeled Con A to the frozen cells revealed no overall loss of binding sites as compared to non-frozen cells although those frozen at 1.7 °C/min appeared to have two types of binding sites whereas the cells frozen at 200 °C/min had only one. Agglutination studies showed that the frozen cells had reduced levels of agglutinability.