A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens.

Cell extracts were used to determine the enzymes involved in the intermediary carbon metabolism of several strains of Shewanella putrefaciens. Enzymes of the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) were detected, but those of the Embden-Meyerhof-Parnas pathway were not. While several tricarboxylic acid cycle enzymes were present under both aerobic and anaerobic conditions, two key enzymes (2-oxoglutarate dehydrogenase and pyruvate dehydrogenase) were greatly diminished under anaerobic conditions. Extracts of cell grown anaerobically on formate as the sole source of carbon and energy were positive for hydroxypyruvate reductase, the key enzyme of the serine pathway in other methylotrophs, while no hexulose synthase activity was seen.     

Gravity-regulated formation of the peg in developing cucumber seedlings

It has been proposed that peg formation in the vascular transition region (TR zone) between the hypocotyl and the root in Cucurbitaceae seedlings is a gravimorphogenetic phenomenon. Initiation of the peg became visible 36 h after imbibition when cucumber (Cucumis sativus L. cv. Burpee Hybrid II) seeds were germinated in a horizontal position at 24°C in the dark. Simultaneously, sedimented amyloplasts (putative statoliths) were apparent in the sheath cells surrounding the vascular strands, and in the cortical cells immediately adjacent to them, in the TR zone. In contrast, the other cortical cells, some of which were destined to develop into the peg, contained amyloplasts which were not sedimented. These results suggest that the graviperception mechanism for peg formation may be like that of statoliths in shoot gravitropism. By 48 h following imbibition, the cells of the TR zone still had sedimented amyloplasts but had lost their sensitivity to gravity, possibly because of their maturation.     

Assessment of xylazine hydrochloride epidural analgesia for open castration of rams

Epidural xylazine injected at the sacrococcygeal site 40 to 150 min prior to surgery (at a dose of 0.05 to 0.10 mg/kg) provided good analgesia during scrotal skin incision in all 20 experimental rams but in only 10 rams (50%) at incision and separation of tunica vaginalis, and 6 rams (30%) during ligation of the spermatic cord. There was a significant correlation between the decrease in heart rate and the dosage of epidural xylazine. Heart rate increased significantly during incision of the tunics and spermatic cord ligation but was not significantly correlated to the clinical assessment of analgesia. There was no significant correlation between the presence of surgical analgesia and the dosage of epidural xylazine: Pelvic limb ataxia was still evident in 12 rams (60%) at 8 h after epidural xylazine injection. Epidural xylazine provided good somatic analgesia during open castration of 20 rams but visceral analgesia was unpredictable. Factors in addition to the dosage of sacrococcygeal epidural xylazine affects the degree of surgical analgesia obtained for open castration of rams.     

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.

The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses.     

Frank Macfarlane Burnet and the immune self

This essay is excerpted from Alfred I. Tauber,The Immune Self: Theory or Metaphor? (New York and Cambridge: Cambridge University Press, 1994).     

Cloning and Expression of a 5-Hydroxytryptamine7 Receptor Positively Coupled to Adenylyl Cyclase

Abstract: In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [¹⁴C]glucose or the [β-³⁵S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.     

The ecological significance of nickel hyperaccumulation: a plant chemical defense

Nickel hyperaccumulating plants have more than 1000 mg Ni kg^–1 dry weight when grown on nickel-bearing soils. We hypothesized that Ni hyperaccumulation could serve as a chemical defense against herbivores In feeding experiments with potential insect herbivores and Ni hyperaccumulating plants, only those inseets fed leaves from plants grown on non-nickel-bearing soil survived or showed a weight gain. Among chemical parameters measured, only Ni content of plants was sufficient to explain this result. When subjected to herbivory by lepidopteran larvae, plants grown on Ni-amended soil showed greater survival and yield than plants on unamended soil. Ni hyperaccumulation may be an effective plant chemical defense against herbivores because of its high lethality, apparent low cost, and broad spectrum of toxicity.     

Effects of trout on the diel periodicity of drifting in baetid mayflies

Some benthic invertebrates in streams make frequent, short journeys downstream in the water column (=drifting). In most streams there are larger numbers of invertebrates in the drift at night than during the day. We tested the hypothesis that nocturnal drifting is a response to avoid predation from fish that feed in the water column during the day. We surveyed diel patterns of drifting by nymphs of the mayfly Baetis coelestis in several streams containing (n=5) and lacking (n=7) populations of rainbow trout, Oncorhynchus mykiss. Drifting was more nocturnal in the presence of trout (85% of daily drift occurred at night) than in their absence (50% of daily drift occurred at night). This shift in periodicity is due to reduced daytime drifting in streams with trout, because at a given nighttime drift density, the daytime drift density of B. coelestis was lower in streams occupied by trout than in troutless streams. Large size classes of B. coelestis were underrepresented in the daytime drift in trout streams compared to nighttime drift in trout streams, and to both day and night drift in troutless streams. Differences in daytime drift density between streams with and without trout were the result of differences in mayfly drift behaviour among streams because predation rates by trout were too low to significantly reduce densities of drifting B. coelestis. We tested for rapid (over 3 days) phenotypic responses to trout presence by adding trout in cages to three of the troutless streams. Nighttime drifting was unaffected by the addition of trout, but daytime drift densities were reduced by 28% below cages containing trout relative to control cages (lacking trout) placed upstream. Drift responses were measured 15 m downstream of the cages suggesting that mayflies detected trout using chemical cues. Overall, these data support the hypothesis that infrequent daytime drifting is an avoidance response to fish that feed in the water column during the day. Avoidance is more pronounced in large individuals and is, at least partially, a phenotypic response mediated by chemical cues.     

The impact of NO inf3 sup− loading on the freshwater macrophyte Littorella uniflora: N utilization strategy in a slow-growing species from oligotrophic habitats

The decline and disappearance of Littorella uniflora from oligotrophic waters which have become eutrophic has been associated with shading or reduced CO₂ supply. However NO _inf3 ^sup– concentrations can reach very high levels (100–2000 mmol m^–3 compared with <1–3 in oligotrophic habitats). To investigate the impact of NO _inf3 ^sup– loading alone, plants were grown under three NO _inf3 ^sup– regimes (very low, near-natural and high). The interactive effects of NO _inf3 ^sup– and photon flux density (low and high regimes) on N assimilation and accumulation, CO₂ concentrating mechanisms, C₃ photosynthesis and growth were also examined. The results were unexpected. Increased NO _inf3 ^sup– supply had very little effect on photosynthetic capacity, crassulacean acid metabolism (CAM) or lacunal CO₂ concentrations ([CO₂]_i), although there was considerable plasticity with respect to light regime. In contrast, increased NO _inf3 ^sup– supply resulted in a marked accumulation of NO _inf3 ^sup– , free amino acids and soluble protein in shoots and roots (up to 25 mol m^–3, 30 mol m^–3 and 9 mg g^–1 fresh weight respectively in roots), while fresh weight and relative growth rate were reduced. Total N content even under the very low NO _inf3 ^sup– regime (1.6–2.3%) was mid-range for aquatic and terrestrial species (and 3.1–4.3% under the high NO _inf3 ^sup– regime). These findings, together with field data, suggest that L. uniflora is not growth limited by low NO _inf3 ^sup– supply in natural oligotophic habitats, due not to an efficient photosynthetic nitrogen use but to a slow growth rate, a low N requirement and to the use of storage to avoid N stress. However the increased NO _inf3 ^sup– concentrations in eutrophic environments seem likely have detrimental effects on the long-term survival of L. uniflora, possibly as a consequence of N accumulation.