Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation.

The comet assay is widely used to measure DNA damage and repair in basic research, genotoxicity testing and human biomonitoring. The conventional format has 1 or 2 gels on a microscope slide, 1 sample per slide. To increase throughput, we have designed and tested a system with 12 smaller gels on one slide, allowing incubation of individual gels with different reagents or enzymes.

Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals.

We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes.

A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division.

In eukaryotes, mitogen-activated protein kinases (MAPKs) are part of signaling modules that transmit diverse stimuli, such as mitogens, developmental cues, or various stresses. Here, we report a novel alfalfa MAPK, Medicago MAP kinase 3 (MMK3). Using an MMK3-specific antibody, we detected the MMK3 protein and its associated activity only in dividing cells.

Characterization of site-directed antisera against endothelin-converting enzymes.

Site-directed antisera have been developed against the two endothelin-converting enzyme-1 (ECE-1) isoforms cloned to date in humans, ECE-1 alpha and ECE-1 beta. Antisera were raised in rabbits against synthetic peptides corresponding to the deduced amino acid sequences that differ between ECE-1 alpha and ECE-1 beta. Antisera were highly selective for their corresponding antigen (titer 1 x 10(4)) and did not detect ET-1 or big ET-1.

Chromatin complexes as aperiodic microcrystalline arrays that regulate genome organisation and expression.

The current understanding of chromatin-mediated repression in Metazoa stems largely from work on two systems in Drosophila: heterochromatin-induced position-effect variegation and repression of the homeotic genes by the Polycomb-group of genes. A common feature of these two systems is the cooperative assembly of multimeric complexes which can epigenetically silence gene activity. Moreover, both older and more recent work has suggested that these complexes can themselves associate to give rise to larger complexes: The specificity of the association is likely to be determined by complementarity of the structural components of the complexes.

Wounding Induces the Rapid and Transient Activation of a Specific MAP Kinase Pathway.

Mechanical injury in plants induces responses that are involved not only in healing but also in defense against a potential pathogen. To understand the intracellular signaling mechanism of wounding, we have investigated the involvement of protein kinases. Using specific antibodies, we showed that wounding alfalfa leaves specifically induces the transient activation of the p44MMK4 kinase, which belongs to the family of mitogen-activated protein kinases.

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants.

Immunocytochemical mapping of a C-terminus anti-peptide antibody to the GABA receptor subunit, RDL in the nervous system in Drosophila melanogaster.

An antibody raised against a peptide based on the C-terminal derived amino acid sequence from a cloned Drosophila melanogaster (fruit fly) gene, Rdl (resistant to dieldrin), was used to investigate localization of a GABA receptor subunit in adult male D. melanogaster. Many regions in the brain and thoracic ganglia were stained with this antibody.

Binding of gelatinases A and B to type-I collagen and other matrix components.

Matrix sequestration of matrix metalloproteinases may be important for the facilitation of remodelling events and the migration of cells through the extracellular matrix. Using an ELISA technique we studied the ability of pro and active forms of gelatinases A and B (GLA and GLB) to bind to matrix components and the contribution made by the different enzyme domains. Pro and active forms of GLA and GLB bound to type-I and type-IV collagens, gelatin and laminin films.

Localization in the nervous system of Drosophila melanogaster of a C-terminus anti-peptide antibody to a cloned Drosophila muscarinic acetylcholine receptor.

Localization in the nervous system of Drosophila melanogaster of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) was investigated using a polyclonal antiserum raised against a peptide corresponding to the predicted receptor carboxyl terminal domain. Immunocytochemical studies on fly sections indicated that the product of the Dm1 mAChR gene was localized in the antennal lobes and in other regions of the brain and thoracic nervous system. Intense staining in the glomeruli of the antennal lobes, the region of the nervous system containing terminals of antennal olfactory sensory neurones and mechanosensory neurones, indicates possible roles for this mAChR gene product in the processing of olfactory and mechanosensory signals in the fly.

Aromatase-immunoreactivity is localised specifically in neurones in the developing mouse hypothalamus and cortex.

Local formation of oestrogens from androgens by aromatase cytochrome P-450 within brain cells is crucial for the sexual differentiation of the mammalian CNS. Aromatase activity has been detected in several brain regions of the developing rodent brain. In the present study, we used a mouse-specific, peptide-generated, polyclonal aromatase antibody to determine whether neurones and/or glial cells in the developing brain are involved in androgen aromatization and if aromatase-immunoreactive (Arom-IR) cells exhibit a sex-specific distribution and regional-specific morphological characteristics.

Aromatase-immunoreactive neurons in the adult female chicken brain detected using a specific antibody.

Estrogen formation in the brain catalysed by the cytochrome P450arom is required for the control of estrogen-dependent neural mechanisms regulating reproductive behaviour. A polyclonal antibody was raised against a 15-amino acid fragment of the chicken ovarian P450arom protein, to localise aromatase-immunoreactive (AR-IR) cells in the adult female chicken brain. Specificity of antibody reaction was established by Western blot and by inhibition of aromatase activity in homogenates of chicken ovarian follicles determined by a radiometric assay.

The porcine insulin-like growth factor-I gene: characterization and expression of alternate transcription sites.

Genomic DNA encoding the 5' region of the porcine IGF-I gene was cloned and sequenced and shown to be highly homologous to that of man, rats and sheep. Two leader exons (exons 1 and 2), which are alternately spliced to exon 3 (encoding part of the mature IGF-I molecule), were identified by RNase protection analysis. In both cases, transcription initiates upstream from exons 1 and 2 at multiple dispersed start sites to yield two distinct IGF-I mRNA transcript classes (1 and 2) which differ in the precursor peptides predicted from their individual leader sequences.

Purification and properties of the Clostridium thermocellum bglB gene product expressed in Escherichia coli.

The Clostridium thermocellum beta-glucosidase B was purified to homogeneity in its recombinant form from Escherichia coli. The purification protocol included ion exchange, hydrophobic interaction and hydroxyapatite chromatography. The polypeptide was found to have a molecular mass of 84,000 daltons and a pI of 4.

Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology.

It is known that two proteins of the cellulosomal complex of Clostridium thermocellum (SL and SS) together degrade crystalline cellulose. SL is a glycoprotein of 210,000 Da which enhances the binding to cellulose and the activity of SS, an endoglucanase of 83,000 Da. We have previously reported the cloning of a DNA fragment encoding the N-terminal end of the SL protein using antibodies raised against the native protein.

Gene sequence and properties of CelI, a family E endoglucanase from Clostridium thermocellum.

The Clostridium thermocellum celI gene, coding for endoglucanase I (CelI), consists of an open reading frame (ORF) of 2640 nucleotides and codes for a protein of M(r) 98531. The ORF was confirmed as celI by comparing the N-terminal sequence of purified recombinant CelI with that deduced from the nucleotide sequence. CelI hydrolysed lichenan and carboxymethylcellulose, but was principally active against barley beta-glucan.

The role of conserved tryptophan residues in the interaction of a bacterial cellulose binding domain with its ligand.

The five conserved tryptophan residues in the cellulose binding domain of xylanase A from Pseudomonas fluorescens subsp. cellulosa were replaced with alanine and phenylalanine. The mutated domains were fused to mature alkaline phosphatase, and the capacity of the hybrid proteins to bind cellulose was assessed.

Localization of immunoreactive endothelin and proendothelin in the human lung.

The endothelins are a family of three 21-amino-acid peptides: endothelin-1, endothelin-2 and endothelin-3. They are powerfully vasoactive, causing both contraction and relaxation of blood vessels. They are also active in the lung causing long lasting bronchoconstriction.

Properties and partial protein sequence of plant annexins.

We have examined the characteristics of Ca(2+)-dependent phospholipid-binding proteins (annexins) in maize (Zea mays L.) coleoptiles and tip-growing pollen tubes of Lilium longiflorum. In maize, there are three such proteins, p35, p33, and p23.

Endothelin-like immunoreactivity in human endometrium.

Endothelin-like immunoreactivity (ET-IR) was detected immunocytochemically in glandular epithelium and vascular endothelium of human endometrium and myometrium. Primary antibody was raised in rabbits against the carboxy-terminal heptapeptide of endothelin 1 (ET-1), ET-1(15-21), and compared with antibodies raised against the cyclized amino-terminal, ET-1(2-13), and commercially obtained antibodies against the whole ET-1 or ET-3 molecule. Binding was visualized using the peroxidase technique in sections counter-stained with haemalum.

Effect of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule.

Short antigenic peptides bound in the groove of class I major histocompatibility complex molecules enable T cells to detect intracellular pathogens. It has been assumed that structural features of the class I molecule alone select which peptides are bound. It is now demonstrated that a complex polymorphism in one of the major histocompatibility complex-encoded putative peptide-transporter genes is associated with an altered spectrum of bound peptides.

Purification and characterization of a new endoglucanase from Clostridium thermocellum.

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.

Localization and endogenous concentration of endothelin-like immunoreactivity in human placenta.

The aim of this study was to investigate the localization of endothelin-like immunoreactivity (ET-IR) in human placenta, chorion and amnion and to compare the endogenous concentration of immunoreactive endothelin (ET) in these tissues before and after the onset of labour. ET-IR was detected in the endothelium of stem vessels in placental villi, as well as in decidual stromal cells in the basal maternal plate, by immunocytochemistry using primary polyclonal rabbit antibody. A specific radioimmunoassay was used to detect endogenous concentration of ET in homogenized placental tissues.

Neuropeptide Y (NPY) metabolism by endopeptidase-2 hinders characterization of NPY receptors in rat kidney.

1. Despite the observation of pharmacological responses to neuropeptide Y (NPY) in mammalian kidneys, there are species differences in the ease with which specific NPY binding sites can be demonstrated; we have investigated whether this can be explained by differential metabolism of NPY by a membrane-bound peptidase. 2.