Thomas L. Lentz

Development of the myotomal neuromuscular junction in Xenopus laevis: An electrophysiological and fine-structural study

The normal development of the myotomal neuromuscular junction in Xenopus embryos and tadpoles was investigated electrophysiologically as well as electron microscopically. Spontaneous potentials, considered to be miniature end-plate potentials (MEPPs), were detected by intracellular recording as early as stage 21 and by stage 24 they were observed in every embryo tested. Like MEPPS at later stages they were blocked by curare but not by tetrodotoxin. End-plate potentials (EPPs), subject to block by tetrodotoxin, were evoked by electrical stimulation of the spinal cord in embryos as young as stage 24 and occurred spontaneously as early as stage 22. The durations of MEPPs and EPPs were initially relatively long. Focal external recordings revealed an eightfold decrease in duration during the course of development. Nerve processes emerged from the spinal cord and contacted developing muscle cells as early as stage 21, but junctional specializations were not apparent and vesicles were rare even in stage 24 embryos. During the next 24 hr, between stages 25 and 36, vesicles increased in number and became localized toward the junctional surface of the nerve ending. Basement lamina developed in the cleft and postjunctional ridges and densities were observed. Individual muscle cells also became contacted by several nerve processes. By stages 48–52 there were fewer contacts on individual muscle cells and Schwann cell processes partially covered the nerve endings. Gap junctions were observed between the muscle cells throughout development but occurred less frequently at the later stages. It is concluded that by the time they reach the muscle cells, or very shortly thereafter, at least some of the growing nerve processes can release transmitter, and some of the muscle cells are sufficiently sensitive to acetylcholine in the region of contact to respond with millivolt depolarizations. These earliest functional contacts, however, are morphologically undifferentiated.

The recognition event between virus and host cell receptor : a target for antiviral agents

Conclusion The initial stage in the viral infectious cycle is the attachment of the virus particle to the host cell membrane. Attachment is mediated by the interaction of a viral surface protein, the VAP, with a normal component of the plasma membrane, of either protein, carbohydrate or lipid, which acts as a virus receptor. Information is accumulating rapidly not only on the nature of the VAPs and of viral receptors but also on the specific domains involved in binding. This knowledge is allowing the development of molecular models for the virus-host cell interaction. Such models permit the rational design of agents which might be effective in preventing virus infection by blocking the attachment stage of the infectious cycle. One group of agents (ligand mimics) will resemble, chemically or structurally, the binding domain of the ligand (in this case the VAP) and competitively inhibit binding of virus to the receptor. The second group of agents will resemble the binding domain of the receptor (receptor mimics) and prevent binding of virus with the host cell by attaching to the binding domain of the VAP.

Tissue Factor (Thromboplastin): Localization to Plasma Membranes by Peroxidase-Conjugated Antibodies

Peroxidase-conjugated antibodies were used to determine the histologic and cytologic localization of bovine and human tissue factor (thromboplastin). Tissue factor antigen was found in highest concentration in the intima of blood vessels, particularly in the plasma membranes of endothelial cells and in human atheromatous plaques. Tissue factor was also found limited to the plasma membranes of many cell types. The presence of tissue factor in the plasma membranes of endothelial cells and atheromata suggests that it may play a significant role in hemostasis and thrombosis.

Nerve Trophic Function: In vitro Assay of Effects of Nerve Tissue on Muscle Cholinesterase Activity

The effects of tissue explants and nerve homogenates on cholinesterase activity of muscle of the newt Triturus in organ culture were measured. Sensory ganglia, ganglia separated from muscle by a Millipore filter, spinal cord, liver, and nerve homogenates produced greater activity of muscle cholinesterase than occurred in untreated muscle cultured for the same period of time. Boiled ganglia, kidney, oviduct, and spleen were ineffective. This procedure serves as a convenient bioassay for a neurotrophic process and indicates that the trophic effect is mediated by a diffusible chemical substance produced by nerves.

Hydra: Induction of Supernumerary Heads by Isolated Neurosecretory Granules

Neurosecretory granules of Hydra littoralis were isolated by differential centrifugation. Excised midsegments of hydra that were exposed to the fraction containing neurosecretory granules developed additional heads at any site, as they regenerated. Hence neurosecretory granules may contain a factot that regulates or stimulates growth in normal and regenerating hydra.

Rhabdite formation in planaria: The role of microtubules

The differentiation of rhabdite-forming gland cells from neoblasts and elaboration of rhabdites in planaria was investigated. Undifferentiated neoblasts containing numerous cytoplasmic ribosomes acquire an extensive rough-surfaced endoplasmic reticulum and elaborate Golgi apparatus. The rhabdite initially appears as a large Golgi saccule containing opaque granules. The rhabdite becomes enclosed by an extensive system of microtubules. Microtubules are oriented in the long axis of the rhabdite and terminate in the vicinity of the Golgi apparatus situated at the apex of the rhabdite. Golgi vesicles and vacuoles are located external to the microtubules, and some fuse with the limiting membrane of the rhabdite. It is suggested that microtubules mediate the migration of vesicles and vacuoles containing secretory material along their length from the Golgi apparatus to the periphery of the rhabdite where they contribute to its growth. Following elaboration of the rhabdite, the cellular organelles undergo regression. The rhabdite-containing cell migrates from the mesenchyme to the epidermal surface.

Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

The fine structure of differentiating interstitial cells in Hydra

SummaryInterstitial cells of hydra are small undifferentiated cells containing an abundance of free ribosomes and few other cytoplasmic organelles. They are capable of differentiating into epitheliomuscular, digestive, glandular, nerve cells, and cnidoblasts. Developing epitheliomuscular and digestive cells acquire bundles of filaments, 50 Å in diameter, which later are incorporated into the muscular processes. Early gland cells develop an elaborate rough-surfaced endoplasmic reticulum and one or more Golgi apparatus. Secretory granules originate in the Golgi region eventually filling the apex of the cell. Neurons are recognized first by the presence of an elaborate Golgi apparatus, absence of a well-developed endoplasmic reticulum, and later the appearance of cytoplasmic processes. The most striking feature of nematocyst formation by cnidoblasts is the presence of a complex distribution system between protein synthesizing rough-surfaced endoplasmic reticulum and the nematocyst. This system consists of connections between cisternae of the endoplasmic reticulum with smooth Golgi vesicles which in turn are connected to minute tubules, 200 Å in diameter. The tubules extend from the Golgi region around the nematocyst finally entering the limiting membrane of the nematocyst. It is suggested that the interstitial cells of hydra represent a model system for the investigation of many aspects of cell differentiation.

Rabies virus entry at the neuromuscular junction in nerve-muscle cocultures.

Early events in rabies virus entry into neurons were investigated in chick spinal cord–muscle cocultures. Rabies virus (CVS strain) was adsorbed to the surface of cells in the cold. At times up to 10 min of warming to 37°C, virus was most intensely localized to dense swellings on the myotube surface. Texas Red‐labeled α‐bungarotoxin, which binds to nicotinic acetylcholine receptors, colocalized precisely with virus at the densities identifying these regions as neuromuscular junctions. Rabies virus also colocalized in the junctions with synapsin I, a marker for synaptic vesicles. The endosome tracers Lucifer Yellow, Texan Red‐dextran, and rhodamine‐wheat germ agglutinin were added to the cultures at the end of the virus adsorption period and the cultures were warmed. At 10 min, rabies virus and tracers colocalized at neuromuscular junctions and nerve terminals. At 30 min, rabies virus and tracers showed more intense fluorescence over nerve fibers and nerve cell bodies. At 60 min, nerve terminals, nerve fibers, and nerve cell bodies showed intense fluorescence and colocalization for rabies virus and tracers. LysoTracker Red, a marker for acidic compartments, colocalized with rabies virus at nerve–muscle contacts. These findings show that in nerve–muscle cocultures, the neuromuscular junction is the major site of entry into neurons. Colocalization of virus and endosome tracers within nerve terminals indicates that virus resides in an early endosome compartment, some of which are acidified. The progressive increase of virus and tracers in nerve fibers and nerve cell bodies over time is consistent with retrograde transport of endocytosed virus from the motor nerve terminal.

Neurotoxin-binding site on the acetylcholine receptor.

Publisher Summary The nicotinic acetylcholine receptor (AChR) at the neuromuscular junction transduces a chemical signal, the neurotransmitter acetylcholine released in response to an action potential at the motor nerve terminal, into an electrical event in the muscle cell that eventually activates the contraction process. In response to acetylcholine, the AChR undergoes a conformational change in which a cation-selective channel is opened, allowing sodium ions to enter and depolarize the cell. Essential functions of the receptor during this process are the binding of acetylcholine to the AChR, the coupling of the binding event to elements of the receptor comprising the channel, the opening of the channel to allow inward passage of cations, and the mechanisms that modulate these steps. Identification of the acetylcholine-binding site on the AChR would increase understanding of the mechanism by which binding of the ligand leads to the changes in the receptor that result in opening of the channel. Localization of this site is greatly facilitated by the use of snake venom curaremimetic neurotoxins, which bind to the receptor with considerably higher affinity than acetylcholine. There is evidence that the toxin-binding site includes the acetylcholine-binding site so that use of the toxins as biological probes should permit characterization of the site on the AChR involved in the binding of acetylcholine. The use of toxins as probes along with techniques employing other ligands, peptide fragments, monoclonal antibodies, synthetic receptor peptides, and genetic engineering are yielding considerable information on the toxin-binding site. In this chapter, current information on the localization of the curaremimetic neurotoxin-binding site on the primary amino acid sequence of the nicotinic AChR is discussed.