Immunogold localization of glucanase-like antifreeze protein in cold acclimated winter rye

Summary Apoplastic antifreeze proteins (AFPs) accumulate in winter rye (Secale cereale L. cv. Musketeer) leaves during cold acclimation. Two of the rye AFPs with molecular masses of 32 and 35 kDa are similar in their amino acid sequences and epitopes to -1, 3-endoglucanase. Localization of these AFPs, which we refer to as glucanase-like proteins (GLPs), was carried out with antiserum raised against the 32 kDa AFP. Specimens from leaves and roots of non-acclimated (NA) plants and cold acclimated (CA) plants were prepared by freeze-substitution for high resolution immunoelectron microscopy. In CA leaves, high levels of GLPs were observed in cell walls of mesophyll cells adjacent to intercellular spaces and in secondary thickenings of xylem vessels. Taken together with the absence of GLPs in vacuoles, these results confirm the apoplastic accumulation of AFPs in CA winter rye. Within the cells of CA leaves, GLPs were localized in cisternae of the rough endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which indicates that GLPs are secreted via an exocytic bulk-flow pathway. The occurrence of high levels of GLPs in CA leaves, their low presence in NA leaves and the lack of GLPs in roots all suggest that there is a correlation between increased accumulation of GLPs and increased freezing tolerance of these plant materials. Furthermore, the localization of GLPs in the immediate vicinity of pathways for free water within the tissues supports the view that these proteins have an important role in the crystallization and/or recrystallization of water when the leaves of CA winter rye are exposed to freezing temperatures.Abbreviations AFP antifreeze protein - BSA bovine serum albumin - CA cold acclimated - GAR goat antirabbit antiserum conjugated with colloidal gold - GLP glucanase-like protein - NA non-acclimated - PBS phosphate buffered saline - PR pathogenesis related     

The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures.

The UL9 protein of herpes simplex virus type 1 (HSV-1) binds specifically to the HSV-1 oriS and oriL origins of replication, and is a DNA helicase and DNA-dependent NTPase. In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments containing oriS. In the absence of ATP, UL9 protein was observed to bind specifically to oriS as a dimer or pair of dimers, which bent the DNA by 35 degrees +/- 15 degrees and 86 degrees +/- 38 degrees respectively, and the DNA was deduced to make a straight line path through the protein complex. In the presence of 4 mM ATP, binding at oriS was enhanced 2-fold, DNA loops or stem-loops were extruded from the UL9 protein complex at oriS, and the DNA in them frequently appeared highly condensed into a tight rod. The stem-loops contained from a few hundred to over one thousand base pairs of DNA and in most, oriS was located at their apex, although in some, oriS was at a border. The DNA in the stem-loops could be stabilized by photocrosslinking, and when Escherichia coli SSB protein was added to the incubations, it bound the stem-loops strongly. Thus the DNA strands in the stem-loops exist in a partially paired, partially single-stranded state presumably making them available for ICP8 binding in vivo. These observations provide direct evidence for an origin specific unwinding by the HSV-1 UL9 protein and for the formation of a relatively stable four-stranded DNA in this process.     

Phosphorylation of mouse thymocyte CD4 and CD8: regulation of surface expression.

Murine T cell surface antigens, CD4 and CD8 are phosphorylated in response to phorbol 12-myristate 13-acetate, a protein kinase C activator, but not phosphorylated after concanavalin A, Ca2+ ionophore or dibutyryl-cAMP treatment. We examined the cell surface expression of both antigens and show that surface CD4 on CD4+CD8+ and CD4+CD8- thymocytes is rapidly decreased after PMA treatment, while CD8 expression is unaffected. Prolonged PMA treatment, which down-regulates protein kinase C, allows CD4 reexpression only in the CD4+CD8- population, suggesting that different mechanisms of cell surface antigen expression are operating in the two thymocyte subpopulations.     

Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.     

Specific deuteration of dichlorobenzoate during reductive dehalogenation by Desulfomonile tiedjei in D2O.

Desulfomonile tiedjei DCB-1 is a strict anaerobe capable of reductively dechlorinating meta-chlorobenzoates. To probe the mechanism of this aryl dechlorination, we incubated cell suspensions of D. tiedjei in D2O and with 2,5-dichlorobenzoate. The deuterium was incorporated into the dechlorination product exclusively at the position of dehalogenation, as shown by gas chromatography-mass spectrometry and proton magnetic resonance analyses. These results favor a model for dechlorination that should not allow proton exchange at other positions, as would be the case if partial ring reduction occurred.     

The effect of packaging films on growth and survival of rhizobia in peat culture

The growth and survival of two strains of rhizobia ( Rhizobium leguminosarum bv. trifolii strain WU95 and R. spp. strain CB3060) injected into finely milled, sterile peat contained in packets of various packaging films were compared after 2, 4 and 8 weeks storage at 26°C. The films were 50 μm and 100 μm low density polyethylene (LDPE), 50 μm high density polyethylene (HDPE) and polyethylene laminated foil and were chosen to provide a range of gas transmission and water permeability properties. Survival of both strains varied directly with the transmission and permeability properties of the film, under controlled storage conditions. These findings provide further evidence that a degree of aeration is necessary for survival of rhizobia in peat-based legume inoculants. The choice of the most suitable film needs consideration of the moisture characteristic curve of the carrier.     

Specific deuteration of dichlorobenzoate during reductive dehalogenation by Desulfomonile tiedjei in D2O.

Desulfomonile tiedjei DCB-1 is a strict anaerobe capable of reductively dechlorinating meta-chlorobenzoates. To probe the mechanism of this aryl dechlorination, we incubated cell suspensions of D. tiedjei in D2O and with 2,5-dichlorobenzoate. The deuterium was incorporated into the dechlorination product exclusively at the position of dehalogenation, as shown by gas chromatography-mass spectrometry and proton magnetic resonance analyses. These results favor a model for dechlorination that should not allow proton exchange at other positions, as would be the case if partial ring reduction occurred.     

N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.