Allen W. Clark

Microtubules in some unicellular glands of two leeches

SummaryWhat appear to be two types of unicellular glands are found in the integument of the leech, Helobdella stagnalis. Type I cells are characterized by a peripheral, subplasmalemmal sack of rough endoplasmic reticulum and accumulations of secretory product in the form of small membrane bound droplets. Type II cells are characterized by large numbers of closely opposed sacks of rough endoplasmic reticulum and secretory product in the form of large, evidently amorphous accumulations of secretory product.Both cell types attenuate into long, slender processes through which the secretory product passes to the surface of the leech. Each process is characterized by a subplasmalemmal sack of ER which runs the entire length of the process and is continuous, at the proximal end of the process, with sacks of rough ER. Associated with the inner member of the ER membrane pair are microtubules with a diameter of approximately 240 Å.A similar arrangement of a subplasmalemmal ER sack associated with microtubules also is found in secretory processes of the leech, Macrobdella decora.The possible source and functions of these microtubules are discussed.

Suppression by elevated calcium of black widow spider venom activity at frog neuromuscular junctions

SummaryFrog neuromuscular junctions were treated with both concentrated black widow spider venom (BWSV) and elevated extracellular calcium (5–50 mM). This procedure causes a dramatic increase in the frequency of spontaneous miniature endplate potentials (mepps) which persists for only a few minutes. In contrast, BWSV-induced mepp activity, the venom effect (VE), continues for 20 min–1 h at junctions in elevated calcium Ringer solutions treated with doses of dilute venom or at junctions in normal calcium (1.91 mM) Ringer solution treated with concentrated venom. Following the disappearance of the VE in elevated extracellular calcium, only a few normal amplitude mepps and a few giant amplitude mepps are observed. The disappearance of the VE in these preparations is irreversible and occurs whether exposure to elevated extracellular calcium precedes or follows exposure to BWSV.Electron microscopy indicates that the major structural alterations produced by exposure to concentrated BWSV and 20 mM calcium Ringer solution are the swelling of nerve terminal mitochondria and the clumping of synaptic vesicles, large numbers of which remain in the terminals. Exposure to 20 mM calcium Ringer solution alone produces no ultrastructural modifications in these preparations.These observations can best be explained if one of the effects of BWSV is to increase the permeability of the nerve terminal membrane to calcium. Only doses of concentrated venom can sufficiently elevate intracellular calcium to a concentration at which synaptic vesicles clump together, thus interrupting the transmitter release process.

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme.

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.

The plantar nerves—lumbrical muscles: a useful nerve—muscle preparation for assaying the effects of botulinum neurotoxin

The plantar nerves-lumbrical muscles (PL) of the hindpaw of the mouse is a preparation that is particularly useful for assaying the effects of botulinum neurotoxin (NT) on the mammalian neuromuscular junction. Each mouse provides a minimum of 4 and a maximum of 8 such preparations. The muscles are thin enough so that neuromuscular junctions can be easily located with Nomarski interference contrast optics for impaling with microelectrodes. The preparations are small enough so they can be sectioned transversely or longitudinally, in their entirety, for light and electron microscopy, or just the regions containing end-plates can be used. An examination of the responses of this preparation to types A and E NT reveal its exceptional suitability for experiments with NT. The small number of fibers and their arrangement permit assays of low concentrations of NT to proceed to complete and total paralysis in a convenient period of time.

Major E. coli heat-stress protein do not translocate: implications for cell survival.

When Escherichia coli are exposed to heat stress, the majority of proteins in the process of synthesis at the time of heat stress are rapidly translocated to the outer membrane of the bacterium. The synthesis of most of these proteins appears to take place on membrane-bound polyribosomes. With the temperature shift, overall protein synthesis is inhibited while the synthesis of a small group of proteins is initiated. These proteins are not translocated, but remain in the cytosolic compartment, and they are identifiable as heat-stress proteins. Both the translocation phenomenon and the retention of heat-stress proteins in the cytosolic compartment in proximity to the nucleoid could counteract the effects of heat stress. The translocated proteins may operate by stabilizing the outer membrane prior to the induction of heat-stress proteins and the latter, which are confined to the cytoplasmic compartment, may serve to protect the integrity of the nucleoid structures.