Cell surface distribution and intracellular fate of human beta-very low density lipoprotein in cultured peritoneal mouse macrophages: a cytochemical and immunocytochemical study

The receptor-mediated endocytosis pathway of colloidal gold labeled beta-very low density lipoprotein (beta-VLDL-Au) derived from patients with familial dysbetalipoproteinemia was analyzed at the ultrastructural level in macrophages. The results showed that beta-VLDL-Au complexes were specifically recognized by a cell surface receptor of the macrophages. beta-VLDL-Au particles once bound to the randomly distributed cell surface receptors clustered in coated pits and were taken up by coated vesicles. Subsequently, the beta-VLDL-Au particles passed through tubular structures and small endosomes before deposited into large electron lucent smooth surfaced endosomes. As revealed by ruthenium red and enzyme cytochemistry the endosomes appeared to be separated from the extracellular space and did not contain acid phosphatase. There were no clear signs of passage of beta-VLDL through the Golgi complex. The accumulation of many flocculated gold particles within Ac-Pase positive vesicles suggests that beta-VLDL once internalized by the macrophages is diverted into a degradative pathway. Incubation of beta-VLDL-loaded macrophages with the hydrophobic fluorescent dye nile red revealed numerous large fluorescent bodies within the cells indicating that the macrophages accumulate large amounts of lipid droplets with time. Additional studies large amounts of lipid droplets with time. Additional studies with native beta-VLDL in conjunction with postembedding immunocytochemical techniques were used to delineate further the intracellular pathway. Immunolabeling was carried out on thin sections of LR White embedded cells using affinity-purified polyclonal rabbit antibodies against apolipoprotein B with the protein A-gold or goat anti-rabbit IgG-gold technique. Indirect visualization of beta-VLDL by these immunocytochemical studies yielded results comparable to those with gold-labeled beta-VLDL. On the basis of both indirect immunocytochemical and direct cytochemical localization of beta-VLDL it is concluded that although colloidal gold labeling of beta-VLDL molecules unquestionably modifies their morphology, their function appears to be unaltered, at least with respect to the process of receptor-mediated endocytosis.     

Acetylcholinesterase activity in antennal receptor neurons of the sphinx moth Manduca sexta

Summary We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the antenna of the sphinx moth Manduca sexta. High levels of echothiophate-insensitive (presumably intracellular) AChE activity were found in six different types of antennal receptors localized in specific regions of the three antennal segments of the adult moth. Mechanosensory organs in the scape and pedicel, the Böhm bristles and Johnston's organ, are innervated by AChE-positive neurons. In each annulus of the antennal flagellum, AChE-positive neurons are associated with six sensilla chaetica and a peg organ, probably a sensillum styloconicum. At least 112 receptor neurons (8–10 per annulus) innervating the intersegmental membranes between the 14 distalmost annuli also exhibit high levels of echothiophate-resistant AChE. In addition, each annulus has more than 30 AChE-positive somata in the epidermis of the scale-covered (back) side of the flagellum, and 4 AChE-positive somata reside within the first annulus of the flagellum. Since none of the olfactory receptor neurons show a high level of echothiophateresistant AChE activity, and all known mechanoreceptors are AChE-positive, apparently intracellular AChE activity in the antenna correlates well with mechanosensory functions and is consistent with the idea that these cells employ acetylcholine as a neurotransmitter.     

Synchrony of bone marrow proliferation and maturation as the origin of cyclic haemopoiesis

Abstract. Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non-cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG-CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.     

Acoustic orientation in adult, female crickets (Gryllus bimaculatus de Geer) after unilateral foreleg amputation in the larva

Summary InGryllus bimaculatus females one foreleg was amputated at the coxa-trochanter joint in the 2nd, 4th or 8th/9th larval instar. A leg of up to normal length is regenerated (Fig. 1) but it lacks a functional ear. In spite of the, usually shorter, regenerated foreleg, the adult one-eared crickets show no impairments in walking when tested on a locomotion compensator. Without sound they walk erratically and most of them weakly circle towards the intact side (Fig. 2).With calling song presentation three response types can be distinguished:tracking (Fig. 3A), hanging on (Fig. 3B) or continuouscircling towards the intact side (Fig. 3C, D). Turning tendencies in monaurals increase with song intensity and exceed those of intact and bilaterally operated animals (Fig. 4). Course deviations towards the intact side also slightly increase with intensity (Fig. 5). Course stability is reduced compared to that of intact animals but exceeds that of bilaterally operated crickets (Figs. 5, 6). It is best at 60 dB and deteriorates at higher sound intensities (Fig. 6). The percentage of monaurals tracking or hanging on decreases with increasing intensity (Fig. 7B). Tracking is established in most animals but it is limited to a narrow intensity range (Fig. 7A, C). Apart from an increased percentage of tracking after early operations (Fig. 7D), there are no prominent changes in orientational parameters with the date of foreleg amputation.Reamputation of the regenerated leg in the adult monaurals does not significantly impair acoustic orientation (Figs. 8, 9), but occlusion of the ipsilateral prothoracic spiracle does (Figs. 10, 11).An attempt is made to correlate the behavioral performance with the activity of auditory interneurons which have undergone morphological and physiological changes (Fig. 12).     

Karyological stability and microevolution of a cell suspension culture ofBrachycome dichromosomatica (Asteraceae)

A long-term suspension culture ofBrachycome dichromosomatica (2n = 4) was induced from a cotyledon-derived callus. Subcultures were obtained every week up to three years. The bulk of the cultures displayed a stable diploid karyotype, while one cell line evolved with 2n = 5 chromosomes in the 86th reinoculation. No further chromosomal change occurred also in that cell line. It is assumed that the fifth chromosome is the expression of a trisomy 2.The chromatin ultrastructure was of the species-specific chromomeric type in the wild-type line, while the trisomic line displayed more condensed chromatin, what probably indicates a rather inactive state of the extra-chromosome.Brachycome dichromosomatica is suggested to represent an ideal species to follow-up karyotype stability and/or variation in cell culture.As a former student W. N. dedicates this paper in gratitude and admiration to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday. Prof.Woess with her scientific work has stimulated in an unique manner the study of nuclear structures in plants, of endopolyploidy and polytene chromosomes, and has thus established the basis for the rapidly increasing research in these fields.     

Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.     

Nerve cell and nematocyte production in Hydra is deregulated by lithium ions

Summary LiCl, a well-known vegetalising agent, interferes with the commitment of stem cells to nerve cells and nematocytes in Hydra attenuata. Treatment with 20 mM LiCl inhibits commitment to nerve cells, treatment with 1 mM LiCl inhibits commitment to nematocytes. However, LiCl does not prevent stem cells committed to the nematocyte pathway from dividing and differentiating into nests of nematocytes. Following LiCl treatment, determination to nerve cells and nematocytes is triggered again. Commitment to nerve cells is strongly stimulated within the first 3 h following pulse treatment with LiCl if the animals have been fed immediately prior to treatment. In Hydra exposed to LiCl for 10 days the stem cell density is reduced by at least 90% of the initial value, and nematocytes are almost completely missing, whereas the density of nerve cells is within the normal range in animals with normal morphology. Animals which developed a transverse constriction in the middle of the body axis contain a 1.7-fold higher nerve cell density in the lower part than is observed in control animals.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

Genetic and blood coagulation characterization of “Swedish” families with von Willebrand's disease types I and III: New aspects of heredity

Summary Twenty-five patients with von Willebrand's disease (vWD) type III were analysed with regard to blood coagulation variables and possible deletions. Nine of the probands and their families were further investigated with DNA linkage analyses. Different patterns of heredity can be suggested in our families with vWD type III, on the basis of blood coagulation analyses. The findings suggest homozygosity in five families and the possibility of compound heterozygosity or a new mutation in the proband in three families. The linkage analyses confirm the results of the coagulation analyses. The segregation of the von Willebrand factor (vWF) gene can be followed in the families, and carrier diagnosis can be made in several of the probands' relatives. The possibility of large deletions in the vWF gene of the probands and their parents was investigated with probes representing the whole vWF cDNA. No deletions were found.This study was approved by the Ethics Committee of the Karolinska Hospital (No. 84:1)