Mannose induces an endonuclease responsible for DNA laddering in plant cells.

The effect of D-mannose (Man) on plant cells was studied in two different systems: Arabidopsis roots and maize (Zea mays) suspension-cultured cells. In both systems, exposure to D-Man was associated with a subset of features characteristic of apoptosis, as assessed by oligonucleosomal fragmentation and microscopy analysis. Furthermore, D-Man induced the release of cytochrome c from mitochondria. The specificity of D-Man was evaluated by comparing the effects of diastereomers such as L-Man, D-glucose, and D-galactose. Of these treatments, only D-Man caused a reduction in final fresh weight with concomitant oligonucleosomal fragmentation. Man-induced DNA laddering coincided with the activation of a DNase in maize cytosolic extracts and with the appearance of single 35-kD band detected using an in-gel DNase assay. The DNase activity was further confirmed by using covalently closed circular plasmid DNA as a substrate. It appears that D-Man, a safe and readily accessible compound, offers remarkable features for the study of apoptosis in plant cells.

SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.

Action of 50 Hz magnetic fields on cyclic AMP and intercellular communication in monolayers and spheroids of mammalian cells.

To investigate the influence of physiological parameters such as cell density and three-dimensional cell contact on the biological action of a 2 mT/50 Hz magnetic field, mouse fibroblasts were exposed as monolayers and as multicellular spheroids. Changes in cyclic AMP content of cells and alterations in gap junction-mediated intercellular communication were measured immediately after 5 min of exposure to the field. In monolayers of intermediate cell density (1 x 10(5) cells/cm2), the field treatment caused an increase in cAMP to 121% of the control level, whereas, at 3 x 10(5) cells/cm2 (near confluence), a decrease to 88% of the unexposed cells was observed. Furthermore, field exposure stimulated gap-junction communication to 160% of the control level as determined by Lucifer yellow dye exchange. In spheroids, alterations in the radial profile of cellular cAMP were observed that were due both to field-induced local cAMP changes and to increased gap-junction permeability for this second messenger, the latter causing radial cAMP gradients to be flattened. The results indicate a strong dependence of field action on physiological parameters of the system exposed.

Signaling the arrest of pollen tube development in self-incompatible plants.

Self-incompatibility (SI), the cellular recognition system that limits inbreeding, has served as a paradigm for the study of cell-to-cell communication in plants since the phenomenon was first described by Darwin. Recent studies indicate that SI is achieved by diverse molecular mechanisms in different plant species. In the mustard family, the mechanism of SI shows parallels to the signaling systems found in animals that are mediated by cell-surface receptors with signal-transducing protein kinase activity.

Capillary oxygen transport during severe hypoxia: role of hemoglobin oxygen affinity.

The efficacy of an increased hemoglobin oxygen affinity [decreased oxygen half-saturation pressure of hemoglobin (P50)] on capillary oxygen transport was evaluated in the hamster retractor muscle under conditions of a severely limited oxygen supply resulting from the combined effects of a 40% reduction in systemic hematocrit and hypoxic ventilation (inspired oxygen fraction 0.1). Two groups of hamsters were utilized: one with a normal oxygen affinity (untreated; P50 = 26.1 +/- 2.4 Torr) and one with an increased oxygen affinity (treated; P50 = 15.7 +/- 1.4 Torr) induced by the chronic short-term administration of sodium cyanate. Using in vivo video microscopy and image analysis techniques, we determined oxygen saturation and associated hemodynamics at both ends of the capillary network. During hypoxic ventilation, the decrease in oxygen saturation across the network was 3.6% for untreated animals compared with 9.9% for treated animals. During hypoxia, estimated end-capillary PO2 was significantly higher in the untreated animals. These data indicate that, at the capillary level, a decreased P50 is advantageous for tissue oxygenation when oxygen supply is severely compromised, because normal oxygen losses in capillaries are maintained in treated but not in untreated animals. The data are consistent with the presence of a diffusion limitation for oxygen during severe hypoxia in animals with a normal hemoglobin oxygen affinity.

Relationship between capillary and systemic venous PO2 during nonhypoxic and hypoxic ventilation.

We evaluated the relationship between end-capillary and systemic venous PO2 values in the retractor muscle of 14 anesthetized hamsters during both nonhypoxic and hypoxic ventilation to ascertain whether the level of tissue oxygenation could be reliably estimated from the systemic parameter. End-capillary PO2 was estimated from measurements of oxygen saturation in capillaries at the venular end of the network obtained using in vivo video microscopy and computer-aided image-analysis techniques at three different levels of inspired oxygen (0.3, 0.21, and 0.1). Measurements of systemic arterial and venous blood gases were made in conjunction with these capillary determinations. In addition, in a portion of the study we utilized an oxygen microelectrode to determine the PO2 in the first-order venule draining the portion of the muscle containing the capillaries under study. We found that only when the animals were made acutely hypoxic was there any correspondence between the systemic venous and end-capillary PO2 values. In addition, these data provide support for the presence of arteriovenous shunting of oxygen during nonhypoxic ventilation.

An alternative transcript of the S locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein.

Recent reports have shown that SLG, one of two genes linked to the S locus of Brassica, encodes a secreted glycoprotein. We have used RNA gel blot analysis, genomic and cDNA clone analysis, expression in transgenic plants, and immunodetection to characterize SLG2, the SLG gene derived from the S2 haplotype. This haplotype belongs to the class II group of S haplotypes that exhibit a weak incompatibility phenotype and are pollen recessive. We showed that SLG2 produces two transcript forms: the expected 1.6-kb transcript that predicts a secreted glycoprotein and an alternative 1.8-kb transcript that predicts a membrane-anchored protein. Stigmas of the S2 haplotype and pistils of transgenic tobacco plants transformed with the SLG2 gene produce a membrane-associated 62-kD protein as well as soluble 57- and 58-kD glycoforms. Because of the sequence similarity between SLG2 and the extracellular domain of the S Locus Receptor Kinase (SRK2) gene, the membrane-anchored form of SLG2 may be viewed as a naturally occurring truncated form of the receptor that lacks the kinase catalytic domain. The occurrence of this protein has potential implications for the activity of the full-length receptor. Furthermore, the underlying structure of the SLG2 gene suggests the evolution of SLG from an ancestral SRK-like gene.

A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L., encodes a functional serine/threonine kinase.

To investigate the catalytic properties of the Brassica oleracea S-locus receptor kinase (SRK), we have expressed the domain that is homologous to protein kinases as a fusion protein in Escherichia coli. Following in vivo labeling of cultures with 32P-labeled inorganic phosphate, we observed phosphorylation of the fusion protein on serine and threonine, but not on tyrosine. In contrast, labeling was not observed when lysine-524, a residue conserved among all protein kinases, was mutated to arginine, thus confirming that SRK phosphorylation was the result of intrinsic serine/threonine kinase activity.

Microvascular oxygen transport: impact of a left-shifted dissociation curve.

The impact of an increased hemoglobin oxygen affinity (decreased P50) on oxygen transport was evaluated in capillaries of the retractor muscle under nonhypoxic (FIo2 = 0.30 and 0.21) and hypoxic (FIo2 = 0.10) conditions in hamsters with normal oxygen affinity [control; P50 = 26.1 +/- 1.0 (SD) mmHg, n = 12] and in hamsters with an increased oxygen affinity [treated; P50 = 16.2 +/- 1.6 (SD) mmHg, n = 7] induced by chronic short-term administration of sodium cyanate. Using in vivo video microscopy and computer-aided image analysis, we determined oxygen saturation (SO2) and associated hemodynamic parameters in both arteriolar (n = 30 control, 18 treated) and venular (n = 25 control, 17 treated) capillaries. In response to hypoxia, systemic arterial PO2 decreased to 29.6 +/- 6.0 (SD) mmHg in control animals and 24.7 +/- 3.8 (SD) mmHg in treated animals associated with abrupt decreases in systemic arterial blood pressure and increases in respiratory rate. The decrease in SO2 across the capillary network during nonhypoxic ventilation was 13.3% SO2 for control animals and 11.0% SO2 for treated animals. During hypoxic ventilation, the decrease in SO2 was 9.1% SO2 in control animals and 8.7% SO2 in treated animals. Hemodynamic parameters were not significantly different in the two groups during hypoxia. Estimated end-capillary PO2 was significantly lower in the treated animals. These data indicate that an increased oxygen affinity does not provide an obvious advantage for oxygen transport during hypoxia at the level of the capillary network in resting striated muscle; however, such an advantage might become apparent in the presence of an increased metabolic rate or a more severe hypoxic challenge.

Retinoic acid modulates gap junctional permeability: a comparative study of dye spreading and ionic coupling in cultured cells.

All-trans retinoic acid (RA), which was recently identified as a morphogen, affects gap junctional permeability in a dose- and time-dependent manner. In five different established mammalian cell lines (FL, BRL, BICR/M1Rk, HEL37, BT5C1) 100 mumol/liter RA reduced Lucifer yellow spreading within 30 min to 20-50% of the control. Ionic coupling, however, remained almost unaffected under the same conditions. Freeze-fractured membranes of untreated and RA-treated cells were similar with regard to frequency and sizes of gap junction plaques. With concentrations of less than 10 mumol/liter RA the dye spreading increased significantly in the human amniotic cell line FL, pointing to a possible modulatory effect of RA on junctional communication.

Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea.

Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). We describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that resides at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus glycoprotein (SLG) gene and is connected via a single pass transmembrane domain to a protein kinase catalytic center. SRK alleles derived from different S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype.

An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart.

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.

Genomic organization and sequence of the Gus-s alpha allele of the murine beta-glucuronidase gene.

The Gus-s alpha allele of the mouse beta-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r alpha regulatory locus. We isolated Gus-s alpha on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of the Gus-s alpha mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s alpha nucleotide sequence with that of human beta-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.

Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes.

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.

Isolation and sequence of a tryptic peptide containing the autophosphorylation site of the regulatory subunit of bovine brain protein kinase II.

The regulatory subunit (RII-B) of bovine brain protein kinase II and the well-characterized regulatory subunit of heart protein kinase II (RII-H) exhibit similar physicochemical properties, but differ significantly in their peptide maps and antigenic determinants. As a starting point for studying structure/function relationships in RII-B and investigating the extent of homology and diversity between RII-B and RII-H, a peptide containing the autophosphorylation site of RII-B has been characterized. The phosphopeptide was rapidly (36 h) purified to homogeneity (yield = 40%) from a tryptic digest of RII-B using three consecutive reverse-phase high performance liquid chromatography steps. A combination of gas-phase microsequencing and solid-phase Edman degradation was used to determine the sequence and to identify the phosphorylated site: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-A la-Glu. RII-B contains a classical phosphorylation site for the catalytic subunit, and the phosphopeptide sequence is homologous to the sequence surrounding the phosphorylation site of RII-H. Fourteen amino acids are identical in the two sequences, and the high net negative charge on the peptide is conserved. However, the peptide from RII-B is alanine-rich and more hydrophobic. Furthermore, five differences between the two functionally related sequences provide direct evidence for the idea that RII-B and RII-H are the products of related but distinct genes.

Tryptic peptide mapping studies on the regulatory subunits of type II protein kinases from cerebral cortex and heart. Evidence for overall structural divergence and differences in the autophosphorylation and cAMP-binding domains.

Regulatory subunits of type II cAMP-dependent protein kinases (RII) (EC 2.7.1.37) from bovine brain and heart exhibit similar physicochemical and functional properties in vitro. However, the two forms of RII are markedly different in their (a) antigenic determinants, (b) cell and tissue distribution, and (c) subcellular localization. This suggests that each of these cAMP-binding proteins may possess some unique structural features. To assess the degree of overall divergence between the primary structures of brain RII and heart RII, tryptic peptides derived from the two proteins were mapped by reverse phase HPLC on a C18 column. When the column effluent was monitored at 280 nm, 15 peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. More complex HPLC profiles were observed by following peptide absorbance at 210 nm, but a similar level of diversity was apparent: 13 brain-RII-specific and 15 heart-RII-specific tryptic peptides were identified and resolved with a gradient (0-50%) of acetonitrile in 0.1% trifluoroacetic acid. In complementary experiments, classical two-dimensional mapping analyses revealed that several 32P-labeled tryptic fragments derived from autophosphorylated and photoaffinity-labeled brain RII were separate and distinct from the 32P-peptides isolated from similarly treated heart RII. The HPLC mapping data document a structural basis for the immunological disparity between brain RII and heart RII and suggest that the two cAMP-binding proteins are different proteins rather than interconvertible forms of a single protein.(ABSTRACT TRUNCATED AT 250 WORDS)