Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis.

beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose. When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed. The enzyme has a sedimentation coefficient of 9.6S. This beta-galactosidase and the one from Escherichia coli are not antigenically related. Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent. beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively. The hydrolase and transgalactosylase activities of the enzyme are similar to those of E. coli beta-galactosidase.     

A cinematographic investigation into the fish's bioelectric breathing signal

The buccal-ventilatory movements of bluegill sunfish, Lepomis macrochirus and fathead minnows Pimephales promelas and strip chart recordings of these movements were filmed simultaneously. The amplitudes of the ventilatory signals were greatest when a fish was perpendicular to the plane of either electrode, and the polarity of a signal corresponded to the fish's direction to one of the electrodes. The most common breathing waveforms for both species reflected a bimodal pattern. These patterns varied in frequency and shifted toward single modality in response to stress. Portions of the recorded waveforms consistently coincided with the period in which a fish was displaying either full opercular flare or closure. Similar occurrences corresponded with mouth positions. Several commonly occurring variations of the ventilatory signal waveforms are presented, including the response of bluegill sunfish to a strong concentration of chlorinated water.     

A comparative study of the proteoglycan of growth cartilage of normal and rachitic chicks.

1. Proteoglycan was isolated from growth cartilage of normal and rachitic chicks. 2. The proteoglycan from normal cartilage showed differences in chemical composition and physical properties from a comparable fraction isolated from bovine nasal cartilage. 3. The proteoglycan from rachitic-chick cartilage was of smaller size than tis normal counterpart, though of similar average chemical composition. 4. Differences between proteoglycan from normal and rachitic cartilages can be explained in terms of limited proteolytic cleavage.     

Binding and internalization of alpha 2-microglobulin by cultured fibroblasts. Effects of monovalent ionophores

Receptor-bound alpha 2-macroglobulin (alpha 2M) undergoes a two-step process in its internalization by cultured fibroblasts. First, the receptor- alpha 2M complexes concentrate in coated pits on the cell surface. Second, the alpha 2M is internalized into endocytic vesicles we have termed receptosomes. Using a variety of monovalent ionophores and inhibitors of ATP synthesis, the present report provides data that discriminates between these two steps. Appearance of alpha 2M-receptor complexes in coated pits occurs at 4 degrees C and is inhibited by primary amines as well as some other drugs and chemical reagents [1, 2]. Internalization of alpha 2M-receptor complexes into receptosomes is inhibited by monovalent ionophores that disrupt proton gradients (monensin, nigericin, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and 3,3',4',5-tetrachlorosalicyanilide), but not the Na+ specific ionophore antamanide or the K+ specific ionophore valinomycin. Using electron microscopy, the proton ionophores appear to interfere with the transfer of alpha 2M from coated pits to receptosomes. Prolonged incubation with monensin in the presence of alpha 2M also decreases the number of alpha 2M receptors on the cell surface, but this did not appear sufficient to account for the extensive inhibition of internalization. Monensin also inhibited the internalization of vesicular stomatitis virus and epidermal growth factor (EGF). Our data suggest that a proton gradient may be necessary for receptor-mediated endocytosis of alpha 2M and some other ligands.     

Cell-free synthesis of mouse mammary tumor virus Pr77 from virion and intracellular mRNA.

Mouse mammary tumor virus (MuMTV) was purified from two cell lines (GR and Mm5MT/c1), and the genomic RNA was isolated and translated in vitro in cell-free systems derived from mouse L cells and rabbit reticulocytes. The major translation product in both systems was a protein with the molecular weight 77,000. Several other products were also detected, among them a 110,000-dalton and in minor amounts a 160,000-dalton protein. All three polypeptides were specifically immunoprecipitated by antiserum raised against the major core protein of MuMTV (p27), but they were not precipitated by antiserum against the virion glycoprotein gp52. Analysis of the in vitro products by tryptic peptide mapping established their relationship to the virion non-glycosylated structural proteins. The 77,000-dalton polypeptide was found to be similar, if not identical, to an analogous precursor isolated from MuMTV-producing cells. Peptide mapping of the 110,000-dalton protein shows that it contains all of the methionine-labeled peptides found in the 77,000-dalton protein plus some additional peptides. We conclude that the products synthesized in vitro from the genomic MuMTV RNA are related to the non-glycosylated virion structural proteins. Polyadenylic acid-containing RNA from MuMTV-producing cells also directed the synthesis of the 77,000-dalton polypeptide in the L-cell system. If this RNA preparation was first fractionated by sucrose gradient centrifugation the 77,000-dalton protein appeared to be synthesized from mRNA with a sedimentation coefficient between 25 and 35S.     

The cellular localization of malonyl-coenzyme A decarboxylase in rat brain

Malonyl-coenzyme A (CoA) decarboxylase (E.C.4.1.1.9) activity in brain is low but steadily increases after birth. The main physiological role of this mitochondrial enzyme is thought to be the stabilization of malonyl-CoA levels which change very little with brain growth. In an effort to visualize malonyl-CoA decarboxylase by immunocytochemistry, and to determine its developmental changes, the enzyme was purified by an efficient small-scale procedure involving isolation of mitochondria, extraction at high ionic strength, isoelectric focusing, column chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme from brain showed the same apparent molecular weight (160 kDa) and was immunoreactive with antisera raised against malonyl-CoA decarboxylase from liver. Immunocytochemistry revealed early and extensive labeling of hepatocytes in rat liver but only delayed visualization in the brain. Most nerve cells of the cerebral cortex and many microglia were stained but the neurons of the cerebellar cortex did not become reactive. Golgi epithelial cells and their processes, the Bergmann glia, also showed reaction product.     

Barley—Botany,production, harvesting,processing, utilization and economics

Barley, probably the oldest cultivated cereal, is widely grown in cooler areas of the world. The annual world production of nearly two and a half billion bushels exceeds that of rye but is less than that of rice, wheat, corn and oats, respectively. Most of the annual 300 million dollar crop of the U.S. is fed to livestock, but about one- third is manufactured into malt.     

Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury

Following central nervous system trauma, diffuse axonal injury and secondary axotomy result from a cascade of cellular alterations including cytoskeletal and mitochondrial disruption. We have examined the link between intracellular changes following mild/moderate axonal stretch injury and secondary axotomy in rat cortical neurons cultured to relative maturity (21 days in vitro). Axon bundles were transiently stretched to a strain level between 103% and 106% using controlled pressurized fluid. Double-immunohistochemical analysis of neurofilaments, neuronal spectrin, alpha-internexin, cytochrome-c, and ubiquitin was conducted at 24-, 48-, 72-, and 96-h postinjury. Stretch injury resulted in delayed cytoskeletal damage, maximal at 48-h postinjury. Accumulation of cytochrome-c and ubiquitin was also evident at 48 h following injury and colocalized to axonal regions of cytoskeletal disruption. Pretreatment of cultures with cyclosporin-A, an inhibitor of calcineurin and the mitochondrial membrane transitional pore, reduced the degree of cytoskeletal damage in stretch-injured axonal bundles. At 48-h postinjury, 20% of untreated cultures demonstrated secondary axotomy, whereas cyclosporin A-treated axon bundles remained intact. By 72-h postinjury, 50% of control preparations and 7% of cyclosporin A-treated axonal bundles had progressed to secondary axotomy, respectively. Statistical analyses demonstrated a significant (p < 0.05) reduction in secondary axotomy between treated and untreated cultures. In summary, these results suggest that cyclosporin-A reduces progressive cytoskeletal damage and secondary axotomy following transient axonal stretch injury in vitro.     

An anthrone-based Kv7.2/7.3 channel blocker with improved properties for the investigation of psychiatric and neurodegenerative disorders

A set of novel Kv7.2/7.3 (KCNQ2/3) channel blockers was synthesized to address several liabilities of the known compounds XE991 (metabolic instability and CYP inhibition) and the clinical compound DMP 543 (acid instability, insolubility, and lipophilicity). Using the anthrone scaffold of the prior channel blockers, alternative heteroarylmethyl substituents were installed via enolate alkylation reactions. Incorporation of a pyridazine and a fluorinated pyridine gave an analog (compound 18, JDP-107) with a promising combination of potency (IC₅₀ = 0.16 μM in a Kv7.2 thallium flux assay), efficacy in a Kv7.2/7.3 patch clamp assay, and drug-like properties.     

Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.