Ultrastructural localization of egg-laying prohormone-related peptides in the atrial gland of Aplysia californica

The atrial gland is an exocrine organ that secretes into the oviduct of Aplysia californica and expresses three homologous genes belonging to the egglaying hormone gene family. Although post-translational processing of the egg-laying hormone precursor in the neuroendocrine bag cells has been examined in detail, relatively little is known about the post-translational processing of egg-laying hormone-related gene products in the atrial gland. A combination of morphologic techniques that included light-microscopic histology and immunocytochemistry, transmission electron microscopy, and immuno-electron microscopy were used to localize egg-laying hormone-related peptides in the atrial gland and to evaluate the characteristic morphology of their secretory cells. Results of these studies showed that there were at least three major types of secretory cells in the atrial gland (types 1–3). Significantly, of these three cell types, only type 1 was immunoreactive to antisera against egg-laying hormone-related precursor peptides. The immunoreactivity studies established that all three egg-laying hormone-related precursor genes are expressed in type-1 cells and indicated that the processing of these precursors also occurs within the secretory granules of this cell type. Evidence was also obtained that proteolytic processing of the egg-laying hormone-related precursors differed significantly from that observed in the bag cells. In contrast to the bag cells, the NH₂-terminal and COOH-terminal products of the egg-laying hormone-related precursors of the atrial gland were not sorted into different types of vesicles.     

Cellular ATP content of heated Chinese hamster ovary cells

Hyperthermia at either 41.5 or 45 degrees C with variable heating times to reduce cell survival up to three orders of magnitude did not decrease significantly cellular ATP content when measured either immediately or up to 7 hr after a heat treatment. Similarly, cellular ATP content was not significantly reduced with step-down heating, precooling prior to hyperthermia, or thermotolerance induction. The data suggest that heat-induced depletion of intracellular ATP content is not a critical factor in the thermal death of cells heated under normal culture conditions.     

Exposure to pretreatment hypothermia as a determinant of heat killing

When Chinese hamster ovary (CHO) cells were exposed to 22 degrees C for 2 hr prior to 42.4 degrees C hyperthermia, neither the shoulder region of the survival curve nor the characteristic development of thermotolerance after 3-4 hr of heating were observed. Absolute cell survival after 4 hr at 42.4 degrees C was decreased by a factor of between 10 and 100 (depending on the rate of heating of nonprecooled controls). Conditioning at 30 degrees C for 2 hr, 26 degrees C for 2 hr, or 22 degrees C for 20 min followed by heating to 42.4 degrees C over 30 min did not result in sensitization. Prolonged (16 hr) conditioning at 30 degrees C, however, increased the cytotoxicity of immediate exposure to 41.4 or 45 degrees C with maximum sensitization to 45 degrees C occurring after 6 hr at 30 degrees C. Both 3- and 18-hr pretreatments at 30 degrees C similarly increased the cytotoxicity of 45-41.5 degrees C step-down heating (D0 = 28 min in precooled versus 40 min in nonprecooled cells).     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

The molluscan cardioactive neuropeptide FMRFamide: Subcellular localization in bivalve ganglia

Ganglia of the marine mollusk Macrocallista nimbosa were pooled, homogenized, and subjected to differential centrifugation. The neuropeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide) was concentrated in the microsomal pellet. When the medium-speed supernatant was centrifuged in a discontinuous sucrose gradient, three separate peaks of activity were detected and identified as acetylcholine, 5-hydroxytryptamine, and FMRFamide. The relative concentration of FMRFamide in each fraction was determined by bioassay and by radioimmunoassay (RIA). Both determinations revealed a peak of peptide in the middle of the sucrose gradient. Electron micrographs of each of the gradient interfaces were analyzed. The interface containing the peak of biological FMRFamide activity was enriched two- to fivefold in neurosecretory granules with a mean diameter of 104 nm and various electron densities. Morphologically similar vesicles were also seen in intact ganglia. These findings support the notion that FMRFamide is a neurosecretory product. But the physiological function of the peptide in bivalve ganglia remains unknown.     

Assembly of tubulin from cultured cells and comparison with the neurotubulin model

Spontaneous microtubule assembly can be obtained in extracts from a variety of cultured cell lines by including glycerol in the assembly buffer. An analysis of the effects of cultured cell extracts on brain tubulin (neurotubulin) assembly has shown that the extracts contain initiation inhibitors whose effects are diminished by glycerol. By using glycerol during the assembly step, cultured cell tubulin can be purified by assembly-dissassembly procedures. The amount of glycerol necessary for significant spontaneous assembly varies from 1–6 M among the different cell lines and is dependent upon their content of inhibitor. Comparison of the assembly products obtained from NA, C₆ and CHO cells at increasing glycerol concentrations shows that glycerol enhances the purification of tubulin and a polypeptide of molecular weight 49,000 daltons in all three systems. These preparations contain a number of other polypeptides, including a group with gel electrophoretic mobilities characteristic of tau-factor, but lack the high molecular weight microtubule-associated proteins (MAPs) which are present in neurotubulin preparations. Phosphocellulose chromatography of NA tubulin removes several proteins from the tubulin and results in a loss of polymerizability. Among three proteins which are completely removed from the inactive tubulin, the most prominent is the 49K protein. This observation and the co-purification of the 49K protein with tubulin through two assembly-disassembly cycles suggest that it is a true MAP. The difference in MAP proteins between brain and tissue culture cells is parallelled by an absence of ring structures in NA tubulin preparations. NA tubulin, however, does form rings when brain MAPs are added. The early steps of NA tubulin assembly differ from those of neurotubulin; no rings are involved, and the first assembly intermediates are straight protofilament bundles. The differences between MAPs from cultured cells and brain and the absence of ring formation in NA tubulin preparations suggest that the assembly model based on neurotubulin is not completely general. A comparison of extracts from CHO cells grown with and without dibutyryl cAMP revealed no differences between the behavior of these extracts in spontaneous tubulin assembly or in mixture experiments with brain tubulin.     

Procedure for testing kinetic models of the photocycle of bacteriorhodopsin.

Given some simple kinetic models of the photocycle of bacteriorhodopsin (bR) and data taken at many wavelengths and under conditions that avoid photoselection and steady-state cycling complications, it is shown how to extract the apparent rate constants and the spectra of the intermediates. Special consideration was given to establishing the range of error of these results. There are many criteria, which we explicitly discuss, that the spectra should satisfy in order that the kinetic model be acceptable. New data for the photocycle of purple membrane fragments in dilute buffer at pH 7.0 has been obtained at 15 measuring wavelengths and four temperatures. The procedure, which can be generalized to more complex models, has been applied to these data to test two kinds of kinetic models: the unidirectional unbranched model and the undirectional model with simple branching straight back to bR from any intermediate. In these models the spectrum of the O intermediate is highly temperature sensitive, even with branching, and/or has two broad maxima. Moreover, the spectrum of the M intermediate has a secondary maximum and two M-like states appear to be required. Thus, neither model satisfies the physical criteria.     

Dilatometric studies of the subtransition in dipalmitoylphosphatidylcholine

The phase transition between the newly discovered low-temperature subgel phase and the gel phase of dipalmitoylphosphatidylcholine has been studied by using dilatometry. Equilibrium measurements show that the subtransition upon heating is centered at 13.5 degrees C, has a dilatometric half-width of 0.6 degree C, and comprises a specific volume change of 0.009 mL/g (about one-fourth the size of the main transition). When the gel phase is cooled, the subtransition does not occur until below 5 degrees C. The rate of formation as a function of incubation temperature for 1 degree C less than TI less than 6 degrees C was determined; it is not well fit by quantitative theories based upon homogeneous nucleation. However, some form of nucleation is present since temperature-jump studies show that once the subgel phase has started to form, it continues to grow in the range 6 degrees C less than TJ less than 12.8 degrees C. Thus, the true transition temperature lies between 12.8 and 13.5 degrees C, but nucleation of the subgel phase is severely retarded above 6 degrees C, leading to the large hysteresis observed upon cooling.     

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.