Permeability of Alkali Metal Cations in Lobster Muscle : A comparison of electrophysiological and osmometric analyses

Single muscle fibers from lobster walking legs are effectively impermeable to Na, but are permeable to K. They shrink in hyperosmotic NaCl; they swell in low NaCl media which are hyposmotic or which are made isosmotic with the addition of KCl. In conformity, the membrane potential is relatively insensitive to changes in external Na, while it responds according to the Nernst relation for changes in external K. When the medium is made isosmotic or hyperosmotic with RbCl the volume and membrane potential changes are of essentially the same magnitudes as those in media enriched with KCl. The time courses for attaining equilibrium are slower, indicating that Rb is less permeant than K. Substitution of CsCl for NaCl (isosmotic condition) produces no change in volume of the muscle fiber. Addition of CsCl (hyperosmotic condition) causes a shrinkage which attains a steady state, as is the case in hyperosmotic NaCl. Osmotically, therefore, Cs appears to be no more permeant than is Na. However, the membrane depolarizes slowly in Cs-enriched media and eventually comes to behave as an ideal Cs electrode. Thus, the electrode properties of the lobster muscle fiber membrane may not depend upon the diffusional relations of the membrane and ions, and the osmotic permeability of the membrane for a given cation may not correspond with the electrophysiologically deduced permeability. Comparative data on the effects of NH₄ and Li are also included and indicate several other degrees of complexity in the cell membrane.     

Properties of a calcium-activated protease in squid axoplasm which selectively degrades neurofilament proteins

Axoplasm extruded from the giant axon of the squid contains Ca²⁺-activated proteases. The protease in the 100,000X g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF₂₀₀), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca²⁺ ion, and no other divalent cation will substitute. The protease requires Ca²⁺ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF₂₀₀ appears to proceed through a 100,000 MW and possibly a 47,000–50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu²⁺ and Fe²⁺, sulphydryl inhibitors, and leupeptin. This specific Ca²⁺-activated protease in squid axoplasm has identical properties to Ca²⁺-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca²⁺-activated protease purified from human platelets effectively degrades squid NF₂₀₀, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins.     

Effect of aeration during cell growth on ketone reactions by immobilized yeast

The effect of aeration during cell growth on the subsequent reduction of 2-hexanone and 2-octanone by yeast cells entrapped in calcium alginate beads was studied. The reactions were conducted using 2-propanol as a sacrificial substrate to regenerate the cofactor NAD(H), and a mixture of (S)- and (R)-alcohols was produced. The use of strictly aerobic conditions when growing the cells resulted in the highest initial reaction rates, as well as the production of only a single product (i.e., the enantiomeric excess of the (S)-alcohols was 1.0). However, initial reaction rates decreased proportionally with fermentation time regardless of whether the yeast were grown aerobically or under both aerobic and anaerobic conditions. The data also suggest that it is the aerobic (or anaerobic) condition, rather than the cell growth phase, which is responsible for the results seen.     

Slice cultures of LHRH neurons in the presence and absence of brainstem and pituitary

Luteinizing hormone releasing hormone (LHRH) neurons from the preoptic area (POA)/hypothalamus of the postnatal rat were cultured for up to 7 weeks using a slice explant roller culture technique. The slices thinned to quasi-monolayers, but maintained organotypic distributions of large numbers of immunocytochemically identifiable LHRH, neurotensin, tyrosine hydroxylase, neurophysin and corticotropin releasing hormone-containing neurons. The distribution, survival and morphology of LHRH cells in co-cultures with brainstem and anterior pituitary was quantitated, and found to be similar to that observed in single cultures. LHRH fibers grew into either pituitary or brainstem tissue, however when all three tissues were co-cultured, LHRH fibers preferentially invaded the pituitary. LH immunoreactive anterior pituitary gonadotropes were maintained only in co-cultures containing POA/hypothalamic slices, and addition of an LHRH antagonist in such cultures, inhibited LH immunoreactivity in the gonadotropes. This slice explant roller culture method effectively maintains the cyto- and chemoarchitecture and functional properties of the LHRH system for long periods in vitro and should provide excellent models for studying the interactive and molecular characteristics of postnatal LHRH neurons.     

Phylogenetic cross-reactivities of monoclonal antibodies produced against rat neurophysin

Previously described mouse monoclonal antibodies against rat neurophysins [Ben-Barak, Y., et al. (1985); Whitnall, M. H., et al. (1985)] were studied here for their cross-reactivities to neurophysins (NPs) from other vertebrate species. Posterior pituitary extracts from various mammals (rat, mouse, cow, human) and lower vertebrates (frog, ratfish) were studied. The monoclonal antibodies displayed several distinct patterns of cross-reactivity to the various species, indicating that the epitopes which they recognized were different. PS 67 bound strongly to rat pituitary extract in solid-phase radioimmunoassay (RIA) but showed no cross-reactivity with extracts from any of the other species tested, including the mouse. PS 36 cross-reacted with mouse and frog extracts but showed almost no cross-reactivity with cow and none to ratfish extracts. PS 41 cross-reacted with mouse, cow, and frog extracts. PS 45 was the most cross-reactive antibody and recognized an antigen in extracts from mouse, cow, frog, and ratfish pituitaries. Electrophoresis of proteins extracted from posterior pituitaries, followed by immunoblot staining with either PS 36 or PS 45, demonstrated that the NP-like molecules within each species are heterogeneous, i.e., more than two bands stained in each species. The frog NP stained by PS 45 was about twice the molecular weight of the mammalian NPs. The possible valve of the PS 45 antibody for future molecular cloning experiments on the arginine vasotocin precursor in lower vertebrates is discussed.