Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene

The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in ¹³C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO₂. β-Carotene, an isoprenoid plant constituent, was depleted in ¹³C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in ¹³C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less ¹³C) than C₃ plant material but when plants are stressed the isotopic composition could vary.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Photosynthesis in intact leaves of C3 plants: Physics,physiology and rate limitations

The instantaneous rate of photosynthetic CO₂ assimilation in C₃ plants has generally been studied in model systems such as isolated chloroplasts and algae. From these studies and from theoretical analyses of gas exchange behavior it is now possible to study the biochemistry of photosynthesis in intact leaves using a combination of methods, most of which are nondestructive. The limitations to the rate of photosynthesis can be divided among three general classes: (1) the supply or utilization of CO₂, (2) the supply or utilization of light, and (3) the supply or utilization of phosphate. The first limitation is most readily studied by determining how the CO₂ assimilation rate varies with the partial pressure of CO₂ inside the leaf. The second limitation can be studied by determining the quantum requirement of photosynthesis. The third limitation is most easily detected as a loss of O₂ sensitivity of photosynthesis. Measurement of fluorescence from intact leaves can give additional information about the various limitations. These methods are all non-destructive and so can be observed repeatedly as the environment of a leaf is changed. In addition, leaves can be quick-frozen and metabolite concentrations then measured to give more information about the limitations to intact leaf photosynthesis rates. In this review the physics and biochemistry of photosynthesis in intact C₃ leaves, and the interface between physiology and photosynthesis—triose phosphate utilization—are discussed.     

Physiological influences on carbon isotope discrimination in huon pine (Lagarostrobos franklinii)

Summary Measurements of the light environment and stomatal and photosynthetic behaviour are reported for Huon Pine (Lagarostrobos franklinii, family Podocarpaceae) in western Tasmanian rainforest. For a variety of microenvironments, these are used in an analysis of stable carbon isotope measurements in the air, and in branch and leaf material, using a model for carbon isotope fractionation in leaves (Farquhar et al. 1982).The major features of ¹³C variations with respect to branch position can be explained in terms of the direct influence of light level acting via the rate of CO₂ assimilation. In addition a relatively constant ¹³C gradient of about 2.6 between leaf tip and branch wood is observed.Alternative explanations are advanced for the tip-towood gradient in ¹³C. If the ¹³C of leaf tips is taken to represent the value for photosynthate, maintenance respiration is proposed as a mechanism for the further fractionation; a significant ¹³C depletion in respired CO₂ is implied which is not supported by indirect measurements of atmospheric isotope ratio. Furthermore, an assumption of significant sampling errors (e.g. related to humidity effects on assimilation) is required to obtain good quantitative prediction of the light influence.If the branch wood ¹³C is taken to represent that of the photosynthate, the tip-to-wood gradient may find an explanation, via the model, in terms of tip tissue comprising carbon from immature cells. Translocation of photosynthate from exposed to shaded branches is then proposed as a means of obtaining quantitative agreement with the predicted light influence.The support provided for the applicability of the Farqunar et al. (1982) model in the field is discussed in the context of the problem of obtaining past global atmospheric CO₂ levels from ¹³C in tree-rings.     

Steady-state room temperature fluorescence and CO2 assimilation rates in intact leaves

Steady-state room temperature variable fluorescence from leaves was measured as a function of CO₂ pressure in Xanthium strumarium L. and Phaseolus vulgaris L. Measurements were made in a range of light intensities, at normal and low O₂ parital pressure and over a range of temperatures. At low CO₂ pressure fluorescence increased with increasing CO₂. At higher CO₂ pressure fluorescence usually decreased with increasing CO₂ but occasionally increased slightly. The transition CO₂ pressure between the responses could be changed by changing light, O₂ pressure, or temperature. This breakpoint in the fluorescence-CO₂ curve was a reliable indicator of the transition between ribulose 1,5-bisphosphate (RuBP) saturated assimilation and RuBP regeneration limited assimilation. The fluorescence signal was not a reliable indicator of O₂-insensitive assimilation in these C₃ species.     

Osmium tetroxide-zinc iodide reactive sites in the photoreceptor cells of the retina of the rat

Summary The osmium tetroxide-zinc iodide fixative of Champy-Maillet has been used to study the rat's retina at the electron microscope level. Electron opaque deposits were observed all along the photoreceptor cells and concentrated in the outer segments of rods and cones and in the nerve endings. In the outer segments that deposits are located in the inter and intra disk spaces as well as between the disk and outer membranes. In the outer plexiform layer reactive sites include synaptic vesicles and mitochondria; other minor reactive sites are described in the inner segment and inner plexiform layer.Electron opaque deposits were not seen if potassium iodide substitutes zinc iodide in the fixative. However, if osmium tetroxide-potassium iodide fixed retinae are immersed in osmium tetroxide-zinc iodide the characteristic electron-dense material is evidenced at those same sites. The effect of other several fixatives were studied with a similar double fixation procedure. Our finding points to the histochemical demonstration of an unidentified component (s) of the retina which shows a striking specificity of localization and which is made evident when zinc iodide is used in the Champy-Maillet mixture.This work has been supported by grants of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and U.S. Air Force AF-AFOSR 67-0963 A.We are greatly indebted to Miss Haydée Agoff and to Mr. Alberto Saenz for their skillful technical assistance.     

Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

Molecular genetic study of the frequency of monosomy 22q11 in DiGeorge syndrome

It is well established that DiGeorge syndrome (DGS) may be associated with monosomy of 22q11-pter. More recently, DNA probes have been used to detect hemizygosity for this region in patients with no visible karyotypic abnormality. However, DGS has also been described in cases where the cytogenetic abnormality does not involve 22q11; for instance, four cases of 10p- have been reported. In this study we have prospectively analyzed patients, by using DNA markers from 22q11, to assess the frequency of 22q11 rearrangements in DGS. Twenty-one of 22 cases had demonstrable hemizygosity for 22q11. Cytogenetic analysis had identified interstitial deletion in 6 of 16 cases tested; in 6 other cases no karyotype was available. When these results are combined with those from our previous studies, 33 of 35 DGS patients had chromosome 22q11 deletions detectable by DNA probes.