ATPases in rabbit erythrocytes: stimulation by HCO3-anion and by Na-cation-plus-K-cation.

A Mg²⁺Na⁺K⁺ATPase was found in a ghost preparation from rabbit erythrocytes, a finding in conflict with previous reports, but in agreement with the known kinetics of cation movements in these cells. However the Mg²⁺Na⁺K⁺ATPase was not inhibited by 10⁻⁴M ouabain, nor by 10⁻⁴M Ca²⁺. The physiological status of this enzyme is discussed. The basic Mg²⁺-ATPase activity in this preparation is also stimulated by HCO₃⁻; it is suggested that the HCO₃⁻-stimulated ATPases reported in a variety of other preparations are not necessarily due to mitochondrial contamination but could well originate from the plasma membrane.     

ACTIVITY OF ALDEHYDE DEHYDROGENASE, ALDEHYDE REDUCTASE, AND ACETYLCHOLINE ESTERASE IN STRITATUM OF RATS BEARING ELECTROLYTIC LESIONS OF THE MEDIAL FOREBRAIN BUNDLE

A number of enzymes have been measured in the striatum of rats in which the dopamine-containing nerve terminals had been unilaterally destroyed by means of unipolar electrolytic lesions of the medial fore-brain bundle. Fourteen and 28 days after such lesions the tyrosine hydroxylase activity of the striatum was reduced to immeasurably low values, but neither aldehyde dehydrogenase, aldehyde reductase, nor acetylcholine esterase was affected when compared with the striatum from the intact side of the same rat or with those from control rats. These results indicate that in the rat the 3 enzymes are not localized with tyrosine hydroxylase, in the dopaminergic nerve terminals of the striatum. This conclusion is supported by a study of the subcellular localization of aldehyde dehydrogenase in rat brain. This enzyme is distributed between the cytosol and the particulate fraction of brain homogenates separated by centrifugal techniques. with no exceptionally high concentration of the enzyme in the synaptosomal fraction. Because neither of the enzymes of post-deaminative catabolism of dopamine is concentrated in the dopaminergic nerve terminals of the striatum of the rat, it is proposed that in this species the amine is not necessarily taken up by the nerve terminals prior to catabolism.     

EFFECTS OF SINUS NERVE STIMULATION ON CAROTID BODY GLOMUS CELLS

The sinus nerve or sympathetic trunk was stimulated unilaterally in one group of adult cats or Syrian hamsters while in another group the sinus nerve or sympathetic trunk was cut unilaterally and the animals were given reserpine. In a third group, atropine was administered prior to sinus nerve stimulation. All tissues were processed for the detection of primary monoamines. The carotid bodies on the operated sides were compared with those on the unoperated sides of the same animal in order to determine if amine depletion occurred following the experimental procedures. After sinus nerve stimulation alone, the density of the granules in the glomus cells was decreased, but changes were not noted in the granules following sympathetic nerve stimulation. Sinus nerve stimulation after atropine administration resulted in no change in granule density. Sinus nerve transection followed by reserpine treatment resulted in a greater decrease in granule density on the unoperated than on the operated side. Transection of the sympathetic components to the carotid body followed by reserpine injections resulted in a decrease in granule density in the glomus cells on both the operated and unoperated sides. These results suggest that the sinus nerve must be intact for reserpine to exert an effect and that the sinus nerve may contain efferent fibers which modulate amine secretion.     

Ultrastructural changes in the cardiomyopathy of dystrophic hamsters and mice

Changes in the ultrastructure of the cardiac muscle cells have been followed in dystrophic mice and hamsters (22-40 weeks of age) and in both species a severe cardiomyopathy accompanies the cellular damage of the skeletal muscle. The degradative changes of the myofilament apparatus of the heart cells and the specific changes in mitochondrial ultrastructure (including swelling, septation and apparent division) are characteristic of the cellular damage of both the dystrophic skeletal muscle and of normal cardiac muscle in which [Ca]i has been experimentally raised, confirming the suggestions that (i) the same gene is responsible for the myopathy of skeletal and cardiac muscle in animal dystrophy and (ii) that changes in [Ca]i are implicated in the degradative changes of muscle cells.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

UL36p is required for efficient transport of membrane-associated herpes simplex virus type 1 along microtubules

Microtubule-mediated anterograde transport is essential for the transport of herpes simplex virus type 1 (HSV-1) along axons, yet little is known regarding the mechanism and the machinery required for this process. Previously, we were able to reconstitute anterograde transport of HSV-1 on microtubules in an in vitro microchamber assay. Here we report that the large tegument protein UL36p is essential for this trafficking. Using a fluorescently labeled UL36 null HSV-1 strain, KΔUL36GFP, we found that it is possible to isolate a membrane-associated population of this virus. Although these viral particles contained normal amounts of tegument proteins VP16, vhs, and VP22, they displayed a 3-log decrease in infectivity and showed a different morphology compared to UL36p-containing virions. Membrane-associated KΔUL36GFP also displayed a slightly decreased binding to microtubules in our microchamber assay and a two-thirds decrease in the frequency of motility. This decrease in binding and motility was restored when UL36p was supplied in trans by a complementing cell line. These findings suggest that UL36p is necessary for HSV-1 anterograde transport.     

AMPK beta subunit targets metabolic stress sensing to glycogen

AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients, as well as hormones, including adiponectin and leptin. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK. Mammalian AMPK is an alphabetagamma heterotrimer with multiple isoforms of each subunit comprising alpha1, alpha2, beta1, beta2, gamma1, gamma2, and gamma3, which have varying tissue and subcellular expression. Mutations in the AMPK gamma subunit cause glycogen storage disease in humans, but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK beta subunit contains a functional glycogen binding domain (beta-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished beta-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK beta1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.     

Taxonomic implications of genome size and pollen colour and vitality for species of Agapanthus L’Héritier (Agapanthaceae)

Nuclear DNA content (2C) and pollen vitality and colour are used as new criteria to investigate all species of the genus Agapanthus LHéritier. The species have the same chromosome number (2n=2x=30), with exception of four triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 22.1–31.6 pg. This implies that the largest genome contains roughly 10¹⁰ more base pairs than the smallest. The species could be divided in two groups based on pollen colour and DNA content: a group with lilac pollen and a DNA content between 22.3 and 24.1 pg containing the species A. campanulatus Leighton, A. caulescens Sprenger and the rarer A. coddii Leighton, and a group with yellow/brownish pollen and a DNA content from 25.2–31.6 pg containing the species A. praecox Willd., A. inapertus Beauv. and A. africanus (L.) Hoffmanns. Four other taxa, recognized by Leighton (1965) are treated as follows: A. comptonii Leighton, has a nuclear DNA content similar to A. praecox and is considered to be a synonym of A. praecox subsp. minimus Leighton. A. walshii L. Bol., has with 31.6 pg the same high amount of DNA as A. africanus from the same area and is therefore renamed as a subspecies (A. africanus subsp. walshii (Leighton) Zonn. & Duncan comb. nov.). The nuclear DNA amounts of A. dyeri Leighton, including the geographically isolated plants from Mozambique, are shown to be identical to A. inapertus. Therefore A. dyeri is considered synonymous with A. inapertus subsp. intermedius Leighton. A. nutans Leighton is identical in DNA content to A. caulescens and is considered to be synonymous with that species. Concluding there are six species: A. campanulatus Leighton, A. caulescens Sprenger, A. coddii Leighton, A. praecox Willd., A. inapertus Beauv. and A. africanus (L.) Hoffmanns. Nuclear DNA content as measured by flow cytometry and pollen colour are shown to be relevant traits to throw light on the relationships between Agapanthus species.     

Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion

Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.