Quantitation of the O(2)-Dependent, CO(2)-Reversible Component of the Postillumination CO(2) Exchange Transient in Tobacco and Maize Leaves

The postillumination transient of CO₂ exchange and its relation to photorespiration has been examined in leaf discs from tobacco (Nicotiana tabacum) and maize (Zea mays). Studies of the transients observed by infrared gas analysis at 1, 21, and 43% O₂ in an open system were extended using the nonsteady state model described previously (Peterson and Ferrandino 1984 Plant Physiol 76: 976-978). Cumulative CO₂ exchange equivalents (i.e. nanomoles CO₂) versus time were derived from the analyzer responses of individual transients. In tobacco (C₃), subtraction of the time course of cumulative CO₂ exchange under photorespiratory conditions (21 or 43% O₂) from that obtained under nonphotorespiratory conditions (1% O₂) revealed the presence of an O₂-dependent and CO₂-reversible component within the first 60 seconds following darkening. This component was absent in maize (C₄) and at low external O₂:CO₂ ratios (i.e. <100) in tobacco. The size of the component in tobacco increased with net photosynthesis as irradiance was increased and was positively associated with inhibition of net photosynthesis by O₂. This relatively simple and rapid method of analysis of the transient is introduced to eliminate some uncertainties associated with estimation of photorespiration based on the maximal rate of postillumination CO₂ evolution. This method also provides a useful and complementary tool for detecting variation in photorespiration.     

The in situ kinetics of dopamine beta-hydroxylase in bovine adrenomedullary chromaffin cells. Intravesicular compartmentation reduces apparent affinity for the cofactor ascorbate

The Km of dopamine beta-hydroxylase for its cofactor, ascorbic acid, was determined in situ in primary cultures of bovine adrenomedullary chromaffin cells and in isolated chromaffin vesicles. A range of intravesicular ascorbate concentrations in chromaffin cell cultures (1.1-31.2 mM) was achieved by varying the number and concentration of ascorbate additions to the culture media. The rate of octopamine synthesis from tyramine displayed a Michaelis-Menten relationship with respect to ascorbate concentration and an apparent Km of dopamine beta-hydroxylase for ascorbate of 15.0 +/- 2.0 mM was determined. In isolated chromaffin vesicles, with an initial intravesicular ascorbate concentration of approximately 10 mM, ascorbate consumption during beta-hydroxylation occurred as a first order process. This indicated that dopamine beta-hydroxylase was not saturated at this initial ascorbate concentration. When isolated chromaffin vesicles were prepared with different intravesicular ascorbate concentrations, the rate of octopamine synthesis displayed a Michaelis-Menten relationship with respect to ascorbate with an apparent Km of 17.0 +/- 5.0 mM. Ascorbate consumption also occurred as a first order process in ascorbate-loaded chromaffin-vesicle ghosts which had initial ascorbate concentrations of approximately 30 mM but which were depleted of other small molecules such as catecholamines. These results indicate that the in situ Km of dopamine beta-hydroxylase for ascorbate (approximately 15 mM) is 25-fold higher than it is for the purified or partially purified enzyme assayed under optimal conditions in vitro (0.6 mM). The factor(s) which decreases the enzyme affinity for ascorbate, relative to in vitro, resides in the chromaffin vesicle interior and is also retained in chromaffin-vesicle ghosts. The mechanism of this effect remains to be determined. The Km value determined in these experiments is close to the estimated intravesicular ascorbate concentration of bovine chromaffin granules in vivo (4), suggesting that the availability of ascorbate could become a factor in regulating the rate of dopamine beta-hydroxylation.     

Multiple effects of tumor necrosis factor on lipoprotein lipase in vivo

A single dose of recombinant murine tumor necrosis factor (TNF) suppressed lipoprotein lipase activity in adipose tissue of fed rats, mice, and guinea pigs for 48 h, even though TNF itself is rapidly metabolized in vivo. Immunoprecipitation of [35S]lipoprotein lipase from fat pads pulse-labeled with [35S]methionine showed a decrease in relative synthesis of the enzyme, which correlated to the decrease in activity. There was no decrease in general protein synthesis and no change in distribution of the enzyme between adipocytes and extracellular locations in the tissue. This is in contrast to fasting in which case there is redistribution of the enzyme within the tissue, decrease in general protein synthesis, but no change in relative synthesis of lipoprotein lipase. TNF did not decrease lipoprotein lipase activity in any tissue other than the adipose but increased the activity in several cases, most markedly in the liver. No [35S]methionine was incorporated into lipoprotein lipase by liver slices from normal or TNF-treated animals. Thus, the increased activity can not be ascribed to enhanced hepatic synthesis of the enzyme. There was an increase in lipoprotein lipase activity in plasma, which correlated to the increase in liver. Thus, TNF suppresses lipoprotein lipase synthesis in adipocytes, but not in other tissues, and has some as yet undefined effect on lipoprotein lipase turnover in extrahepatic tissues, which results in increased transport of active lipase through plasma to the liver.     

Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia

The ciliate protozoan Paramecium tetraurelia produces secretory granules (trichocysts) which release needle-like structures composed of small, acidic proteins. Using antibodies against isolated chromogranin A (CGA) and against trichocyst proteins, we found cross-reactive proteins in chromaffin granules and trichocysts. Four independently derived sera against isolated CGA stained bands of the Mr 15,000-25,000 family of trichocyst proteins on immunoblots. A positive response was also obtained with antiserum against chemically synthesized peptides (PL26 and GE25) corresponding to defined regions of the CGA amino acid sequence. In extracts of whole Paramecium, larger proteins (Mr 53,000 and 49,000) also reacted with antibodies against CGA and the related synthetic peptides. These larger proteins may represent unprocessed precursors to the smaller proteins of mature trichocysts. Antiserum to trichocysts recognized CGA in chromaffin granule lysates. Further evidence of a Paramecium protein related to CGA was provided by hybridization of Paramecium mRNA with cloned cDNA for bovine CGA. Our results suggest striking conservation in evolution of CGA-like proteins that may play some role, as yet unknown, in secretion.     

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize

Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.     

Transgenic HLA-DR alpha faithfully reconstitutes IE-controlled immune functions and induces cross-tolerance to E alpha in E alpha 0 mutant mice

We have constructed transgenic mice that express the human class II MHC molecule HLA-DR alpha on a genetic background in which the equivalent endogenous gene, H-2 IE alpha, is not expressed. In these mice, DR alpha complemented the E beta chain such that tissue-specific expression of an interspecies hybrid DR alpha-E beta heterodimer was obtained. Despite 25% amino acid differences between DR alpha and E alpha, immune responsiveness to IE-controlled antigens, clonal deletion of IE-reactive T cells, and alloantigenicity were quantitatively and qualitatively indistinguishable in IE-positive mice and in mice that had integrated at least four copies of the transgene. These results demonstrate a remarkable degree of structural, regulatory, and functional conservation. They also suggest that tolerance induction involves only discrete portions of MHC molecules.     

Early and late effects in the bone marrow of mice following 2 Gy (6 MeV) neutron irradiation

Summary Following 5 Gy gamma irradiation, residual damage in bone marrow persisted up to one year and was ascribed to genetic defects in hemopoietic stem cells (von Wangenheim et al. 1986). To see whether high LET radiation is more efficient in inducing late effects, mice were whole-body irradiated with a single dose of 2 Gy neutrons ( = 6 MeV) and femoral cellularity, CFU-S number, proliferation ability of bone marrow cells (PF) and the compartment ratio (CR), i.e. the splenic 125-iodo-deoxyuridine incorporation per transfused CFU-S were measured up to one year after the radiation insult. Within 12 weeks, femoral cellularity, PF and CR recovered to control or near-control level, whereas CFU-S numbers remained significantly below control. No further recovery was observed. On the contrary, PF and CR deteriorated again after 12 and 26 weeks, respectively. CFU-S per femur tended to decrease as well. Thus it is demonstrated that a single dose of 2 Gy 6 MeV neutrons causes significant injury in function (PF) and structure (CFU-S numbers, CR) of bone marrow which persisted up to one year. While this residual injury can be attributed to genetic defects in hemopoietic stem cells, its increasing expression is probably due to late evolving damage in microenvironmental cells. The RBE of 6 MeV neutrons for the introduction of late effects in the bone marrow is in the range of 3.     

Invariant chain--a regulator of antigen presentation

MHC class II molecules present internalized antigens to the immune system. They have long been known to associate with a polypeptide called the invariant chain. Recent findings have revealed that this polypeptide performs two functions. First, it prevents class II molecules from binding antigenic peptides at the site of synthesis of class II molecules in the endoplasmic reticulum.Second, it targets class II molecules to their destination in the endocytic pathway, where they pick up antigenic peptides derived from endocytosed antigens. Short sequences in the cytoplasmic portion of the invariant chain serve as subcellular address labels.The functions of the invariant chain help to explain how the immune system divides its defence against foreign pathogens between cytotoxic T cells and antibodies.