Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

Urinary estrogen excretion during pregnancy in the gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus) and the human (Homo sapiens)

Urinary estrogen components were separated, identified and quantified throughout the pregnancy of the gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) and compared to estrogen levels in normal human pregnancies. Fetal and neonatal adrenals from each species were also compared in terms of weight and relative amounts of fetal zone. The results demonstrate that gorillas and chimpanzees excrete 4- to 5-fold less estrogen during pregnancy than the human and orangutan which are similar to each other. The lower estrogen excretion appears to be related to a smaller fetal adrenal in both the gorilla and chimpanzee which reveal both a reduced adrenal weight and increased definitive to fetal zone ratio when compared to either the human or orangutan.     

Phosphorylation-associated conformation shift of anti-oncogene phosphoprotein p53 in concanavalin-A activated human T lymphocytes.

A new radioimmunoassay for p53, employing an anti-peptide antibody directed against conserved Domain V, exhibited specificity for a relatively dephosphorylated form of p53. This form, correlated with the monoclonal antibody PAB421+ conformation, appeared transiently in the cytosol of cycloheximide-treated T cells undergoing activation by concanavalin-A/serum. Concurrently, there were decreased levels of p53 in the nucleus that correlated with increased phosphorylation of p53. After 90 min nuclear levels of p53 increased steadily above levels of unstimulated cells. Anti-p53 antibodies introduced into cells prior to stimulation enhanced cell proliferation in response to mitogens.     

Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration of spatial distribution of viability

Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 muM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. (c) 1993 John Wiley & Sons, Inc.     

Immunohistochemical localization of articular cartilage proteoglycan and link protein in situ using monoclonal antibodies and lectin-binding methods

Lectins have specificity for certain carbohydrate structures in macromolecules. Lectins are, therefore, useful histochemical tools for demonstrating the composition and localization of components of connective tissue matrices, such as articular cartilage. In order to assess the significance of observed lectin-binding patterns, experiments were performed in which monoclonal antibodies against chondroitin sulphate- and keratan sulphate-containing proteolgycans and link proteins were applied to sections of bovine articular cartilage after enzymatic digestion with chondroitinase ABC and keratanase. The following conclusions were made: (1) Binding of peanut agglutinin (PNA) in the interterritorial matrix predominantly indicates the presence of keratan sulphate, but may also detectO-linked oligosaccharides of proteoglycans. (2) In normal cartilage wheat germ agglutinin (WGA) binds nearly exclusively to keratan sulphate. In cartilage degraded with chondroitinase ABC and keratanase this lectin may also detect carbohydrates in link protein due to enhanced accessibility. Binding of WGA toO-linked oligosaccharides may eventually occur. (3) In enzymatically digested cartilage matrix, staining with soybean agglutinin (SBA) may be due to link protein, but not to chondroitin sulphate, because specific breakdown of the glycosaminoglycan chain is required for binding of SBA. (4)Ulex europaeus agglutinin I (UEA I) binding sites are only detectable in digested cartilage matrix.     

First chromosome scale genomes of ithomiine butterflies (Nymphalidae: Ithomiini): Comparative models for mimicry genetic studies

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Müllerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Recent studies on the ecology and genetics of speciation in Ithomiini have suggested that sexual pheromones, colour pattern and perhaps hostplant could drive reproductive isolation. However, no reference genome was available for Ithomiini, which has hindered further exploration on the genetic architecture of these candidate traits, and more generally on the genomic patterns of divergence. Here, we generated high-quality, chromosome-scale genome assemblies for two Melinaea species, M. marsaeus and M. menophilus, and a draft genome of the species Ithomia salapia. We obtained genomes with a size ranging from 396 to 503 Mb across the three species and scaffold N50 of 40.5 and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. An annotation of transposable elements and gene content was performed, as well as a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models such as Heliconius, and will enable further understanding of the mechanisms of adaptation in butterflies.     

Effects of UV-B on flavonoids, ferulic acid, growth and photosynthesis in barley primary leaves

Barley (Hordeum vulgare L.) was grown with UV-B (280–320 nm) at levels simulating 25 nr 5% ozone depletion on the date of the summer solstice al 40°N latitude, with UV-A (320–400 nm), or with no supplemental irradiation. In plant growth chambers providing 300 μmol m^?2 s^?1 photosynthetically active radiation (PAR). UV-B-grown leaves elongated more slowly than controls but reached the same final length 1 day later. Leal specific fresh weight (mass leaf area^?1) was significantly increased by UV-B after the 7th day of growth. IV-B did not significantly affect leaf area, fresh weight, dry weight, total chlorophylls, total carotenoids or photosynthetic quantum efficiency. CO₂ assimilation was decreased by UV-B only at internal CO₂ levels above 250 μl l^?1. By the 8th day of growth, UV-B increased flavonoid (saponarin and lutonarin) accumulation in both the lower epidermis and the mesophyll: about 40% of the saponarin and 20% of the lutonarin were in the lower epidermis under all experimental conditions. Glasshouse conditions proved too variable for reproducible determination of growth and photosynthesis but were reliable for determining developmental changes in flavonoid (saponarin and lutonarin) accumulation and provided up to 800 μmol m^?2 s^?1 PAR. In the glasshouse UV-B-grown leaves had more flavonoids than controls al all stages from 5 to 30 days after planting: ca 509 more saponarin and 100% more lutonarin. Levels of soluble (vacuolar) ferulic acid esters were similar under all conditions on day 5. and on day 20 or later, but were significantly higher in UV-B-grown plants on days 10 and 15. UV-B decreased insoluble (cell-wall-bound) ferulic acid esters on a whole leaf basis but significantly increased this fraction in the lower epidermis. UV-A had no significant effects on growth, photosynthesis or ferulic acid, but it slightly increased flavonoid accumulation. The results are discussed in terms of secondary phenolics as a tissue-specific, developmentally regulated adaptive response to UV-B.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

RAPID AXOPLASMIC TRANSPORT IN DYSTROPHIC MICE

Abstract— Rapid axoplasmic transport was studied in dystrophic mice of the 129/ReJ-dy strain. Proteins transported in vivo through α-motoneurons of the sciatic nerve were labeled by injections of [³H] or [³⁵S] amino acids into the ventral horn of the lumbar spinal cord. Following an 18 h incubation, axoplasmic transport was quantitated by summing the radioactivity in the 10 mm length of sciatic nerve proximal to a ligation. Although the amount of transported radioactivity was small, transport appeared depressed when adult dystrophic mice were compared to controls. Transport was also studied in the sensory fibers of the sciatic nerve under in vitro conditions, resulting in high levels of transported radioactivity. In this system transport was strongly depressed. The severity of the deficiency varied with age, being small in animals with early clinical signs and becoming maximal (80–90%) in animals over 60 days of age. Proteins transported by adult dy/dy and +/+ animals were compared by gel electrophoresis using double-label techniques. Transport of nearly all proteins was depressed in dy/dy mice, although the possibility exists that small differences occur. The data suggest that the dystrophic state produces a significant deficiency in rapid axoplasmic transport in both motor and sensory fibers, and may interfere with transport processes in all neurons. Since rapid axoplasmic transport has been associated with membranes, the data are consistent with a general alteration of cellular membranes in dystrophic animals.     

A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase.

We describe a new method for quantitatively assaying the omega subunit of Escherichia coli RNA polymerase. The assay is based on the ability of RNA polymerase holoenzyme to catalyze the continuous synthesis of the dinucleotide pApU on a poly[d(A-T)] . poly[d(A-T)] template when supplied with AMP and UTP as substrates. Core enzyme, lacking omega subunit, catalyzed this reaction at a rate less than 1% that of holoenzyme. The omega subunit was not released from the enzyme/DNA complex during dinucleotide synthesis. Using this assay, a titration of a fixed concentration of core enzyme was observed with increasing concentrations of added omega subunit. Below a 1:1 omega:core ratio the measured activity increased linearly with omega concentration, whereas above a 1:1 ratio the activity remained constant. An immediate application of the assay is in determining the concentration of active omega, or equivalently of active holoenzyme, in any RNA polymerase preparation.