ULTRASTRUCTURE AND PERMEABILITY OF NUCLEAR MEMBRANES

The fine structures of nuclear envelopes known to have different permeability properties were compared. Membranes of salivary gland cell nuclei of Drosophila (third instar) and Chironomus (prepupae), which are strong barriers to ion diffusion, and membranes of oocyte nuclei (germinal vesicle) of Xenopus and Triturus, which are much more ion-permeable, show no essential difference in size, frequency, and distribution of their membrane gaps ("pores") which could account for the marked disparities in membrane permeability. The gaps are occupied by diffuse electron-opaque material with occasional central regions of strong opacity. This material may possibly account for the high diffusion resistance of Drosophila and Chironomus nuclear envelopes, where the resistance is far too great to allow free diffusion through the gaps. But material of this kind is also present in the more permeable nuclear envelopes of Xenopus and Triturus oocytes, and there are no convincing structural differences discernible with the techniques employed.     

STUDIES ON AN EPITHELIAL (GLAND) CELL JUNCTION : II. Surface Structure

The surface structure of a gland epithelium (Drosophila salivary gland), particularly that at the junction between cells, was examined under the electron microscope. The junctional surface, which in the preceding paper was shown to be highly permeable to ions, has the following structural characteristics. About two-thirds of it are profusely infolded; the surface membranes of adjoining cells interdigitate and present desmosomes. The width of the intercellular space varies considerably. The remainder of the junctional surface, the third that abuts on the lumen, is rather straight. Here, the cell membranes are aligned parallel at a distance of 150 A, and interconnected at regular intervals of 100 A. The connecting material has a high electron opacity, and is about as thick as the cell membranes, but, unlike the latter, has no resolvable unit membrane structure. The surface at the cell base, which in the preceding paper was shown to be rather impermeable, is infolded and resembles the infolded junctional region. The luminal surface exhibits microvilli. Critical surface dimensions are given, and the implications of surface structure in intercellular permeability are discussed.     

Effects of Temperature on the Generator and Action Potentials of a Sense Organ

Charge transfer through the receptor membrane of the nonmyelinated ending of Pacinian corpuscles is markedly affected by temperature. The rate of rise and the amplitude of the generator potential in response to a constant mechanical stimulus increase with temperature coefficients of 2.5 and 2.0 respectively. The duration of the falling phase, presumably a purely passive component, and the rise time of the generator potential are but little affected by temperature. The following interpretation is offered: Mechanical stimulation causes the conductance of the receptor membrane to increase and ions to flow along their electrochemical gradients. An energy barrier of about 16,000 cal/mole limits the conductance change. The latter increases, thus, steeply with temperature, causing both the rate of rise and the intensity of the generator current to increase. The membrane of the adjacent Ranvier node behaves in a distinctly different manner. The amplitude of the nodal action potential is little changed over a wide range of temperature, while the durations of its rising and falling phases increase markedly. The electrical threshold of the nodal membrane is rather constant between 40 and 12°C. Below 12°C the threshold rises, and the mechanically elicited generator current fails to meet the threshold requirements of the first node. Cold block of nerve impulse initiation then ensues, although the receptor membrane still continues to produce generator potentials in response to mechanical stimulation.     

Effects of Polarization of the Receptor Membrane and of the First Ranvier Node in a Sense Organ

It has previously been shown that the site of production of the generator potential in Pacinian corpuscles is the receptor membrane of the non-myelinated ending, and the site of initiation of the nerve impulse, the adjacent (first) Ranvier node. Effects of membrane polarization of these sites were studied in the present work. Nerve ending and first Ranvier node were isolated by dissection, electric activity was recorded from, and polarizing currents were passed through them. All observations were done at steady levels of polarization, seconds after onset of current flow. The following results were obtained: The amount of charge transferred through the excited receptor membrane is a function of the electrical gradients across the membrane. The generator potential in response to equal mechanical stimuli increases with resting potential of the receptor membrane. The refractory state of the generator potential is not affected by polarization. The electrical threshold for impulse firing at the first Ranvier node (measured by the minimal amplitude of generator potential which elicits a nodal impulse) is nearly minimal at normal resting potential of the node. Both, hyperpolarization and depolarization lead to a rise in nodal threshold. For any level of polarization of nodal and receptor membrane, the threshold for production of impulses by adequate (mechanical) stimulation appears determined by the generator potential-stimulus strength relation and by the electrical threshold of the node.     

Hypothalamic regulation of adiposity: the role of 11beta-hydroxysteroid dehydrogenase type 1.

Following extensive suprasellar operations for excision of hypothalamic tumors, some patients develop morbid obesity despite receiving replacement doses of glucocorticoids. Urine analysis of cortisol and cortisone metabolites show that 11-OH/11-oxo ratios are significantly higher in patients with hypothalamic obesity, indicating enhanced 11beta-HSD1 activity. This correlates with the visceral-to-subcutaneous fat ratio. The consequence of increased 11beta-HSD1 activity and a shift of the steroid inter-conversion towards cortisol may contribute to the effects of the latter in adipose tissue. The message from the hypothalamus to adipocyte 11beta-HSD-1 involves hormones, the sympathetic nervous system and cytokines. CRH and ACTH downregulate 11beta-HSD-1 activity and induce lipolysis. Tumor necrosis factor-alpha and interleukin-1beta upregulate 11beta-HSD-1 expression and activity, while enhancing lipolysis. The sympathetic nervous system exerts its effects through beta-adrenergic upregulation and alpha-adrenergic downregulation of 11beta-HSD-1 activity. Inhibition of 11beta-HSD-1 suppresses preadipocyte differentiation into mature adipocytes, and may provide a therapeutic tool.     

Tyrosine phosphorylation of a gap junction protein correlates with inhibition of cell-to-cell communication

Cell-to-cell communication is achieved by passage of small molecules through gap junction membrane channels. The expression of the transforming gene from Rous sarcoma virus, v-src, induces a rapid and dramatic reduction in cell-to-cell communication in cultured cells. To determine whether connexin43, a major gap junction protein expressed in fibroblasts, is a target for the v-src protein tyrosine kinase activity, we examined the phosphorylation state of connexin43 in cells expressing variants of src. Using an antipeptide serum that recognizes connexin43, we demonstrate that this protein is phosphorylated on serine and tyrosine residues in avian and mammalian cells expressing activated src proteins. Connexin43 from control cells and cells expressing nonactivated variants of the src protein was phosphorylated solely on serine residues. In lysates from v-src-transformed cells, all phosphorylated connexin43 molecules were cleared from the lysate by sequential immunoprecipitations using the phosphotyrosine antibodies, suggesting that each molecule of phosphorylated connexin43 contains both phosphoserine and phosphotyrosine. We have also examined junctional permeability in cells expressing src variants and find that loss of cell-to-cell communication correlates with tyrosine phosphorylation of connexin43.     

Molecular cloning of an oncogenic replication-competent virus that causes lymphoproliferative disease in turkeys

The lymphoproliferative disease virus of turkeys was molecularly cloned, structurally mapped, and shown to represent a distinct class of retroviruses evolutionarily related to the avian leukemia-sarcoma virus group. The cloned provirus did not contain any known oncogene or other cellularly derived sequences and was established as a replication-competent oncogenic entity capable of inducing the disease in the absence of any associated transforming counterpart.     

Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes

Activation of the signal transduction pathways mediated by protein kinase A (PKA) or protein kinase C (PKC) led to different responses of several serum inducible genes including the jun gene family, c-fos, c-myc, krox 20 and krox 24. Whereas all of these genes were stimulated by the phorbol ester TPA, a chemical activator of protein kinase C, they were differently regulated upon cAMP stimulation of the PKA dependent pathway. The proto-oncogenes jun B, c-fos, and to a lesser extent jun D were stimulated by increasing the intracellular concentration of cAMP, whereas the TPA stimulation of c-jun and c-myc was inhibited under these conditions. Krox 20 and krox 24 were insensitive to this second messenger. This study allowed us to classify these growth stimulated genes into three distinct groups distinguished by their sensitivity to elevated concentrations of intracellular cAMP. The inhibition of c-jun and c-myc expression in the presence of increased cAMP levels may be at least partially responsible for the growth inhibitory effect of this agent in Balb/c-3T3 cells.     

The effect of backbone cyclization on PK/PD properties of bioactive peptide-peptoid hybrids: The melanocortin agonist paradigm

A peptide–peptoid hybrid (peptomer) library was designed and synthesized, based on the sequence Phe-d-Phe-Arg-Trp-Gly. This sequence was previously found to specifically activate the melanocortin-4-receptor (MC4R) which participates in regulation of energy homeostasis and appetite. The library of peptomers included a peptoid bond in the Phe and/or d-Phe position and consisted of linear and backbone cyclic analogs, differed in their ring size. While the linear peptides rapidly degraded in serum and in brush border membrane vesicles (BBMV’s), the linear peptomers were more stable. Backbone cyclic peptomers were also stable under the same conditions. Backbone cyclization significantly increased the intestinal permeability of two peptomers compared to their linear counterparts, in the Caco-2 model. Pharmacological evaluation revealed that two linear and one backbone cyclic peptomer, were found active towards MC4R at the nanomolar range. Thus, the conformational constrains imposed by these local and global modifications affect both the pharmacokinetic and pharmacodynamic properties of the parent peptide. This study demonstrates the potential of imposing backbone cyclization on bioactive peptomers as a promising approach in developing an orally available peptidomimetic drug leads.