Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.     

Digestion of prey in foraminifera is not anomalous: A correlation of light microscopic, cytochemical, and hvem technics to study phagotrophy in two allogromiids

Correlative light, high-voltage electron and conventional electron microscopic methods were used to investigate digestion in two allogromiid foraminiferans, Allogromia sp., strain NF, and A. laticollaris Arnold. Microscopic observations showed that bacterial prey are phagocytosed by reticulopodia and are transported to the allogromiid cell body within blister-like phagosomes. Larger prey (algae, diatoms) are transported along the reticulopodial surface and are either stored extrathalamously or phagocytosed at the oral opening (peduncle). Studies of allogromiids optimally fixed and labeled with an extracellular-space label (colloidal thorium) showed that phagocytosed prey are completely enclosed by a plasma membrane envelope; this finding was corroborated by a serial-section three-dimensional reconstruction of the oral zone of one allogromiid. Cytochemical staining for acid phosphatase showed that lysosomes are absent from reticulopods but abundant in the cell body, particularly in the oral zone cytoplasm. We conclude that digestion in allogromiid foraminiferans is accomplished by a vacuole-based digestive apparatus and not by extracellular digestion within a lacunary system, as has been suggested in earlier studies.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5–15 cm H₂O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: (1) Cells exposed to control (3 mm H₂O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H₂O for 7 days. (2) Conditioned medium from endothelial cells exposed to 10 cm H₂O for 7 days contained at ansferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. (3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure. © 1993 Wiley-Liss, Inc.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Surface transport properties of reticulopodia: do intracellular and extracellular motility share a common mechanism?

The reticulopodial networks of the foraminiferan protozoans Allogromia sp., strain NF, and A. laticollaris display rapid (up to 11 microns/second) and bidirectional saltatory transport of membrane surface markers (polystyrene microspheres). Electron microscopy shows that microspheres adhere directly to the reticulopodial surface glycocalyx. A videomicroscopic analysis of this phenomenon reveals that microsphere movement is typically independent of pseudopod extension/withdrawal and that particles of different sizes and surface properties display similar motile characteristics. The motile properties of surface-associated microspheres appear identical to those of saltating intracellular organelles. Indeed, in some instances the surface-attached microspheres appear transiently linked in motion to these underlying organelles. Our observations suggest that, in reticulopodia, surface transport of microspheres and intracellular transport of organelles are driven by a common mechanism.     

Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.     

A preliminary report of vibriosis in cultured American lobsters,Homarus americanus

A vibrio-like bacterium, designated Vibrio sp. (BML 79-078), was isolated from moribund juvenile Amercan lobsters, Homarus americanus. This is the first report of a vibrio-like bacterium being associated with a disease of lobsters. Koch's postulates have been satisfied for this bacterium. In this study, Vibrio sp. (BML 79-078) and Vibrio anguillarum (ATCC 19264) were found to be pathogenic when injected into juvenile lobsters held at 20°C, while Enterobacter aerogenes was not. An endotoxin was found to be associated with the pathogenesis of the disease syndrome caused by Vibrio sp. (BML 79-087).     

Mode of action of somatostatin in inhibiting parathyroid hormone secretion

Effects of somatostatin on basal and low calcium-, isoproterenol- or dibutryl cyclic AMP (DBcAMP)-stimulated parathyroid hormone (PTH) secretion were evaluated in vitro with bovine parathyroid tissue. Low calcium, isoproterenol or DBcAMP alone significantly stimulated PTH secretion. Somatostatin 1 or 4 microgram/ml significantly inhibited these stimulated PTH secretions. Inhibition of isoproterenol-stimulated PTH secretion was more complete than was the inhibition of low calcium- or DBcAMP-stimulated secretion. The studies indicate that somatostatin inhibits PTH secretion by an action distal to cAMP generation. The more complete inhibition of isoproterenol-stimulated PTH secretion suggests that somatostatin may also have additional effects on or proximal to the formation of cyclic AMP.     

Absence of Functional Leptin Receptor Isoforms in the POUND (Leprdb/lb) Mouse Is Associated with Muscle Atrophy and Altered Myoblast Proliferation and Differentiation

Objective

Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr^db/lb) to determine the role of leptin in skeletal muscle.

Methods and Findings

Skeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30–40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.

Conclusion

These data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.