Mitogens for murine embryo cell lines

The growth-promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2α, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T1/2). Each cell had an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2α were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.     

The activation of bovine Factor X by bovine Factor Xa

A steady-state kinetic analysis of the activation of bovine Factor X, by bovine Factor X_a, was undertaken. The activation was found to be dependent on the presence of divalent cations; Ca²⁺ showing the greatest stimulatory effect and Mn²⁺ exhibiting a lower degree of activity for this reaction. Although Sr²⁺ and Mg²⁺ were ineffective when present alone, each contributed synergistically to the activation rate at suboptimal levels of Ca²⁺. The effect of phospholipid (phosphatidylcholine:phosphatidylserine, 4:1, w:w) on the rate of activation and on the activation pathway was investigated. Phospholipid (PL) concentrations of up to 40 μm had no effect on the activation rate; whereas, concentrations of 40–180 μm were slightly inhibitory. In the absence of PL, the major product of the activation was Factor α-X_a, while in the presence of PL, lower-molecular-weight forms of Factor X (Factor β-X) and Factor X_a (Factor β-X_a were produced. At saturating levels of Ca²⁺, the K_{m app} for the activation, at pH 7.4 and 37 °C, in the absence of PL, was found to be 0.6 ± 0.1 μm and the V was 1.7 ± 0.3 mol Factor X cleaved min^?1 mol^?1 Factor X_a. The corresponding values, in the presence of 90 μm PL, were 1.4 ± 0.2 μm and 2.2 ± 0.2 mol Factor X cleaved min^?1 mol^?1 Factor X_a.     

Two distinct regulatory steps in cartilage differentiation

The effect of developmental stage on chondrogenic capacity in high-density cell cultures of chick embryonic wing bud mesenchyme is examined. Mesenchyme from stage 19 embryos forms aggregates of closely associated cells which do not form cartilage matrix, nor contain significant levels of type II collagen that are detectable by immunofluorescence, unless they are treated with dibutyryl cyclic AMP. Mesenchyme from stage 24 embryonic wing buds in high-density cell cultures will spontaneously form cartilage, as defined by electron microscopy and immunofluorescence with antibody to type II collagen. Cultures prepared from stage 26 wings form numerous aggregates which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells morphologically. However, because these cells show considerable intracellular immunofluorescence for type II collagen, they are actually unexpressed cartilage cells. Several treatments, including exposure to dibutyryl cyclic AMP, ascorbic acid and an atmosphere of 5% oxygen, or mixture with small numbers of stage 24 wing mesenchyme cells, stimulate expression, as determined by the accumulation of an Alcian blue-staining matrix and an ultrastructurally recognizable cartilage matrix. Since the addition of similar numbers of differentiated cartilage cells does not stimulate expression of stage 26 cells, it is proposed that initial cartilage expression is dependent on a mesenchyme-specific influence which might be removed by cell dissociation. These studies demonstrate that there are at least two distinct transitions in cartilage differentiation: one involves the conversion of mesenchyme to unexpressed chondrocytes and the second involves mesenchyme-dependent expression of chondrogenic differentiation.     

Mitogenic property of the apical ectodermal ridge

While the apical ectodermal ridge (AER) is well known for its required role in the development of distal parts of the limb and for its ability to stimulate limb duplications, the mechanism of its action is unknown. In this study we use a culture system previously developed by M. Globus and S. Vethamany-Globus (1976, Differentiation6, 91–96) in which an AER grafted onto a high-density cell culture of limb mesenchyme stimulates the formation of an outgrowth. Time-lapse movies taken during the outgrowth period demonstrated no cellular activities other than cell division. Both the mitotic index and labeling index in the mesenchyme were significantly elevated under the AER as compared to that without AER, indicating that the AER provides a growth-promoting stimulus which increases the proportion of dividing cells. On the other hand, nonridge ectoderm had no detectable effect on the mitotic index. Treatment of cultures with cytosine arabinoside both inhibited DNA synthesis and prevented AER-induced outgrowth. These results demonstrate a mitogenic capacity of AER tissue and suggest that mesenchymal outgrowth requires this activity. The mitogenic property of the AER is considered in relation to limb outgrowth in situ.     

The Friesner Herbarium (BUT) of Butler University

The Friesner Herbarium (BUT) of Butler University is a collection of over 100,000 specimens built from the personal herbarium of Ray C. Friesner. He and other botanists at Butler amassed one of the largest and most complete collections of Indiana plants. Active exchange from the 1920’s through the 1940’s increased the holdings of plants from other states. Although the collection does not contain many type specimens, it is rich in vouchers from floristic and ecological studies conducted in the first half of the 20th century and published in the scientific journal,Butler University Botanical Studies.     

Endopeptidase-24.11, a Cell-Surface Peptidace of Central Nervous System Neurons, Is Expressed by Schwann Cells in the Pig Peripheral Nervous System

Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and cervical sympathetic trunk in which greater than 99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed 125I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM. Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelin- and non-myelin-forming Schwann cells. Endopeptidase-24.11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.     

Epithelial differentiation in the absence of extracellular matrix

Summary To investigate the regulation of epithelial differentiation, normal human epidermal keratinocytes were cultured floating on the surface of culture medium without attachment to a solid substrate. Keratinocytes spread out on the surface of the medium, proliferated and differentiated either into several flat lacy sheets 1 to 3 cells thick (on medium containing 0.15 mM calcium) or formed one single aggregate of cells from 5 to 15 cells in thickness on medium containing 1.15 mM calcium. The cell aggregates demonstrated a pattern of ordered epithelial differentiation. Levels of progressive differentiation resembling the structure of normal human epidermis were identified by light microscopy, immunohistochemistry, and electron microscopy. Differentiation proceeded from cells at the air side toward cells at the medium side with basal appearing cells on the air side and keratinocytes expressing filaggrin and involucrin on the side toward the medium. These results demonstrate that organized epithelial differentiation can occur in the absence of extracellular matrix. In contrast with other air-liquid interface cultures, epithelial differentiation in the absence of extracellular matrix progresses from air towards medium.     

Electron-Microscopic Demonstration of a “Head and Stalk” Structure of the Leaf Vacuolar ATPase in Mesembryanthemum crystallinum L.

The structure of the vacuolar ATPase from mesophyll tonoplasts of Mesembryanthemum crystallinum has been studied by electron microscopy using negatively stained specimens of membrane-bound and detergent-solubilized ATPase molecules. We observed a high density of particles on the surface of tonoplast vesicles and “head and stalk” structures on the edge of the membrane, similar to the F₀F₁-ATPases of mitochondrial and chloroplast membranes. The staining conditions, which are often critical for such small objects, were improved by using methylamine tungstate as negative stain for the membrane-bound ATPase. Compared to other staining solutions generally applied, dissociation of the F₁-like enzyme complex from the membrane was best prevented and structural damage of the vesicles was least observed with methylamine tungstate. In freeze-fracture electron microscopy of tonoplast vesicles, where dissociation never occurs since no detergent is used, we also observed “head and stalk” structures on the edge of the membranes, beside many particles on the fracture faces. The detergent-solubilized ATPase forms string-like structures, caused by the aggregation of the hydrophobic membrane-embedded F₀-like part of the enzyme. After negative staining the F₁-like enzyme complex is arranged alternately along both sides of the string and connected by a narrow stalk.     

Nutritional requirements for conidial germination and hyphal growth of Beauveria bassiana

Nutritional requirements for germination and growth of the entomopathogenic fungus Beauveria bassiana are not complex. For germination to occur, a utilizable source of carbon must be present; however, a nitrogen source is needed for continued hyphal growth, otherwise lysis ensues. Compounds that can serve as utilizable carbon-energy sources for germination include glucose, N-acetylglucosamine, glucosamine, chitin, starch, lanolin, hydrocarbons in crude oil, and some longer-chain fatty acids. Both organic and inorganic sources of nitrogen are readily utilized for growth. Conidia undergo active metabolism soon after being placed in a suitable growth medium, indicating that conidia are released from their state of dormancy several hours before emergence of the germ tube can be observed. Because of the nutritional versatility of B. bassiana, this fungus should be able to survive and be infective in several types of natural environments.