Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA). A free-Ca²⁺ activity of less than 40 M restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca²⁺-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca²⁺ during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca²⁺ addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N,-tetracetic acid - FR far-red light - Mes 2-(N-morpholino)ethanesulphonic, acid - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos.

Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities.     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

Energy metabolism and selective neuronal vulnerability following global cerebral ischemia

A short period of global ischemia results in the death of selected subpopulations of neurons. Some advances have been made in understanding events which might contribute to the selectivity of this damage but the cellular changes which culminate in neuronal death remain poorly defined. This overview examines the metabolic state of tissue in the post-ischemic period and the relationship of changes to the development of damage in areas containing ischemia-susceptible neurons. During early recirculation there is substantial recovery of ATP, phosphocreatine and related metabolites in all brain regions. However, this recovery does not signal restitution of normal energy metabolism as reductions of the oxidative metabolism of glucose are seen in many areas and may persist for several days. Furthermore, decreases in pyruvate-supported respiration develop in mitochondria from at least one ischemia-susceptible region at times coincident with the earliest histological evidence of ischemia-induced degeneration. These mitochondrial changes could simply be an early marker of irreversible damage but the available evidence is equally consistent with these contributing to the degenerative process and offering a potential site for therapeutic intervention.Submitted as an Overview article for the volume of Neurochemical Research in honor of Alan N. Davison.     

Photoinhibition of holly (Ilex aquifolium) in the field during the winter

The distribution of holly ( Ilex aquifolium ) and its habitat preferences indicate a sensitivity to low temperature, particularly when exposed to high light. Experiments were conducted to determine whether photoinhibition of photosynthesis occurs in holly leaves in the field in United Kingdom during the winter. Photosynthetic efficiency was assessed in holly leaves that were exposed to or shaded from direct sunlight using measurements of photosynthetic oxygen evolution and chlorophyll fluorescence. Field measurements were conducted over 3 weeks during January and February. Correlation of the measurements of photosynthetic efficiency with weather conditions indicated that holly was suffering photoinhibition, particularly in leaves exposed to direct sunlight. Controlled environment studies demonstrated that exposure of leaves to low temperature and high light resulted in reductions in photosynthetic efficiency; however, leaves recovered rapidly when exposed to a higher temperature and reduced light level.     

Developmental Regulation of the (1,3)-beta-Glucan (Callose) Synthase from Tomato : Possible Role of Endogenous Phospholipases

Activity levels of UDP-glucose: (1,3)-β-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit (Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1-phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A₂, C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action.     

Biosynthesis of gallotannins

Cell-free extracts from leaves of Rhus typhina L. (sumach) were found to transfer the 1-O-galloyl moiety of l,6-di-O-galloyl-β-d-glucose to the 2-position of the same compound, yielding 1,2,6-tri-O-galloyl-β-d-glucose and leaving 6-O-galloylglucose as the deacylated by-product. The enzyme catalyzing this ‘disproportionation’ was purified almost 1700-fold. It had a molecular weight of approx. 56 000, a K _m value of 11.5 mM, was stable between pH 4.5 and 6.5, and most active at pH 5.9 and 40° C. The systematic name “1,6-di-O-galloyl-glucose: 1,6-di-O-galloylglucose 2-O-galloyltransferase” (EC 2.3.1.) was proposed for this new enzyme whose detection provided evidence that, in addition to β-glucogallin (1-O-galloyl-β-d-glucose), higher substituted glucose esters also have the potential to serve as acyl donors in the biosynthesis of gallotannins.     

Phytoalexins of woody plants

Summary Phytoalexins accumulated in selected woody plants in response to microbial attack or stress are reviewed and listed with respect to their chemical structure and probable biogenetic origin. The host-pathogen systems from which they have been isolated are described. The review also considers the antimicrobial activity of the phytoalexins to the causal pathogens and other microorganisms.     

Face recognition.

The study of face-selective neurons in the monkey temporal lobe, and face recognition deficits in humans after brain damage have both become very active fields of investigation. Face-selective neurons appear to be members of ensembles for coding faces rather than individual face detectors or grandmother cells. They reflect the more general role of temporal cortex in pattern recognition. In humans there are a variety of face-processing impairments that result from damage to different areas, and which reflect interference at different levels of processing of the facial image.