The envelope anion channel involved in chloroplast protein import is associated with Tic110.

An anion channel of the chloroplast envelope was previously shown to be involved in protein import. Some gating characteristics of the channel are presented. The pore size of the channel is estimated to be around 6.5 A. Antibodies raised to Tic110 completely inactivate the protein import-related channel. These observations suggest that the channel is associated with the Tic machinery and can function as the protein conducting channel of the inner envelope membrane.     

Monitoring the Metabolic State of Fungal Hyphae and the Presence of Melanin by Nonlinear Spectral Imaging

Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of Agaricus bisporus (white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.     

Expression,purification, and characterization of the intra-cellular domain of the ANP receptor

The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCA_ID comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn²⁺ or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn²⁺ requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases.     

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria.

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30 degrees C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23 degrees C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.     

Cell-surface heparan sulfate and heparan-sulfate/chondroitin-sulfate hybrid proteoglycans of mouse mammary epithelial cells

The hydrophobic cell-surface proteoglycans of mouse mammary epithelial cells were purified by gel filtration, ion-exchange chromatography, and liposome incorporation. The size of the proteoglycans appeared to be directly proportional to the size of their heparan-sulfate chains, larger proteoglycans yielding larger chains. The chondroitin sulfate chains, in contrast, showed no size heterogeneity. Digestion of 125I-labeled proteoglycans with heparitin-sulfate lyase and chondroitin ABC lyase yielded core proteins of approximately 93 kDa, approximately 85 kDa and approximately 38 kDa. Comparison with single enzyme digestions identified the 93-kDa and 85-kDa cores as components of hybrid proteoglycans that carried both heparan-sulfate and chondroitin-sulfate chains. Immunoblotting indicated that the 93-kDa and 85-kDa cores shared the epitope defined by monoclonal antibody 281-2. The 38-kDa core, in contrast, carried only heparan-sulfate chains and lacked the 281-2 epitope. Preparations enriched in heparan sulfate or in heparan-sulfate/chondroitin-sulfate hybrid proteoglycans were obtained by N-desulfation and ion-exchange chromatography. Hybrid proteoglycans accounting for the bulk of the chondroitin-sulfate and nearly half of the heparan-sulfate residues of the proteoglycans showed a similar polydispersity of heparan-sulfate chain sizes as found in proteoglycans that carried only, or predominantly, heparan-sulfate chains. These hybrids contained heparan-sulfate and chondroitin-sulfate chains in similar molar amounts. Analysis of 125I-labeled proteoglycans suggested that typical hybrid proteoglycans were composed of a 85-kDa core protein that carries a single chondroitin-sulfate chain and a single heparan-sulfate chain of variable length. A minority of hybrids seemed characterized by the variant, but possibly structurally related, 93-kDa core protein. The other half of the hydrophobic proteoglycans were composed of the 38-kDa core and carried only heparan-sulfate chains. The significance of the co-existence of hybrid and heparan-sulfate proteoglycans at the cell surface and possible relationships between the proteoglycans need to be further clarified.     

De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells

Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple copies and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK- mutants which are the result of plasmid loss. Under these conditions TK- clones were isolated with a frequency of 5.10(-6) both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK- derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK- variants. In the TK- derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK- variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of the two single-copy TK+ cell lines no TK- mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK- again at a 100-fold higher rate than the original TK+ cell line.