Hydroxyurea-induced partial mushroom body ablation does not affect acquisition and retention of olfactory differential conditioning in honeybees

The mushroom bodies (MBs), a paired structure in the insect brain, play a major role in storing and retrieving olfactory memories. We tested whether olfactory learning and odor processing is impaired in honeybees in which MB subunits were partially ablated. Using hydroxyurea (HU) to selectively kill proliferating cells, we created honeybees with varying degrees of MB lesions. Three-dimensional reconstructions of brains were generated to analyze the drug-induced morphological changes. These reconstructions show that, with few exceptions, only the MBs were affected by the drug, while other brain areas remained morphometrically intact. Typically, lesions affected only the MB in one hemisphere of the brain. To preclude HU-induced physiologic deficits in the antennal lobe (AL) affecting olfactory learning, we measured the responses to odors in the AL using an in vivo calcium imaging approach. The response patterns did not differ between the AL of intact versus ablated brain sides within respective specimens. We, therefore, carried out side-specific classical discriminative olfactory conditioning of the proboscis extension reflex (PER) with control bees and with HU-treated bees with or without MB ablations. All experimental groups learned equally to discriminate and respond to a rewarded (CS+) but not to an unrewarded (CS-) conditioned stimulus during acquisition and retention tests. Thus, our results indicate that partial MB lesions do not affect this form of elemental olfactory learning.     

Male mate choice and sperm allocation in a sexual/asexual mating complex of Poecilia (Poeciliidae, Teleostei)

Male mate choice is critical for understanding the evolution and maintenance of sexual/asexual mating complexes involving sperm-dependent, gynogenetic species. Amazon mollies (Poecilia formosa) require sperm to trigger embryogenesis, but the males (e.g. Poecilia mexicana) do not contribute genes. Males benefit from mating with Amazon mollies, because such matings make males more attractive to conspecific females, but they might control the cost of such matings by providing less sperm to Amazon mollies. We examined this at the behavioural and sperm levels. P. mexicana males preferred to mate with, and transferred more sperm to conspecific females. However, if males mated with P. formosa, sperm was readily transferred. This underscores the importance of male choice in this system.     

Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum

In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Andrenocorticotropin and β-lipotropin in the hypothalamus

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.     

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications

Background

The regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards.