Electron microprobe analysis of intracellular electrolytes in resting and isoproterenol-stimulated exocrine glands of frog skin

Summary In the intact, in vitro frog skin, isoproterenol (ISO) stimulates and amiloride-insensitive increase in short-circuit current (SCC) that can be localized to the exocrine glands and is associated with secretion of chloride. To determine which cells in the glands respond to stimulation we measured the intracellular electrolyte concentrations of the various cell types of the mucous and seromucous glands of the skin using freeze-dried cryosections and electron microprobe analysis. In the resting state, the various cell types of the glands have intracellular electrolyte concentrations similar to the epithelial cells of the skin. Exposure to amiloride (10^–4 m) has little effect on the concentration of Na and Cl in the cells of the glands. The effect of isoproterenol has two distinct phases. Analysis of glands in tissues frozen at the peak of the SCC response (13 min after addition of isoproterenol) shows that the only significant change is an increase in Na and Ca in a group of cells at the ductal pole of the acini of both gland types. These are termed gland cells. The duct cells and cells that secrete macromolecules did not show any significant changes at this timepoint. In the gland cells, after a one-hour exposure to isoproterenol the Na concentration is at prestimulation levels while Cl drops. There is also a smaller drop in Cl in the duct and skin epithelial cells. Ouabain, which can completely block the isoproterenol SCC response, has little short-term effect on Na and Cl in the control gland but accentuates the gain of Na and drop in Cl in the isoproterenol-treated condition. Bumetanide and, to a lesser extent, furosemide, also blocks the isoproterenol SCC response and causes a further drop in Cl. The results provide indirect evidence that a major portion of the ionic component of the gland secretion is produced by a distinct group of cells separate from those producing the macromolecular component and that the mechanism of secretion involves a Na:Cl coupled transport system linked to the activity of the basolateral Na pump.     

GTPγS restores nucleophosmin (NPM) localization to nucleoli of GTP-depleted HeLa cells

Previous studies showed that localization of nucleophosmin/B23 (NPM) to nucleoli requires adequate cellular GTP levels (Finchet al., J Biol Chem 268, 5823–5827, 1993). In order to study whether hydrolysis of GTP plays a role in NPM localization, we introduced a nonhydrolyzable GTP analog into HeLa cells. Cells were first depleted of GTP with the IMP dehydrogenase inhibitor, mycophenolic acid (MA), to induce translocation of NPM from the nucleoli to the nucleoplasm. Non-hydrolyzable GTP analogs were then introduced into cells by electroporation. We found that introduction of the non-hydrolyzable analog, GTPS, was effective in restoring NPM localization to nucleoli. Cells incubated in medium containing G-nucleotides without electroporation showed no effect. To reduce the possibility that cells use guanine from degraded nucleotide to supplement GTP pools via salvage pathways, experiments were also performed in the presence of (6-mercaptopurine) 6MP, a competitive inhibitor of the salvage enzyme, HGPRT (hypoxanthine guanine phosphoribosyl transferase), in addition to MA. Under these conditions, introduction of GTPS still effectively restored the localization of NPM into nucleoli. This study demonstrates that electroporation can be used effectively to introduce nucleotides into cultured cells without excessive loss of viability. Our results also indicate that the GTP dependent localization of NPM to the nucleoli may not require GTP hydrolysis.     

Genetics of esterases in species of Lycopersicon

Summary Improvements in plant culture and electrophoretic technique permit detection and genetic analysis of seven esterase loci in Lycopersicon esculentum and related species with homosequential chromosomes. At all of these loci except one, each allele codes for a single anodal band, and the electrophoretic variants are inherited in monogenic fashion. For the exceptional Est-4, allozymes are 1–3 banded in various combinations at four positions, and rare recombinants in one cross appeared at a frequency of 0.0005, suggesting the existence of several very tightly linked genes. Est-2 segregated solely for intensity differences in dominant/recessive fashion; Est-3 and Est-4 behave as monomers; the remaining Est-l, 5, 6, and 7 — coding for contiguous bands in the region closest to the origin — are dimeric. The latter group are tightly linked inter se in the proximal portion of 2L (long arm of chromosome 2), the total map distance of the complex being approximately 1.5 cM; Est-2 is situated on 9L between ah and marm; Est-3 on 1L between inv and dgt; Est-4 has not yet been located. Even in the interspecific hybrids, map distances are similar to the standard values for L. esculentum. Tandem duplication is hypothesized for the origin of the Est-l, 5–7 complex, which adds another example to the growing list of linked mimic genes in the tomato genome. In respect to the position of their bands and tight inter se linkage, this series exactly parallels the EA, EB, EC esterase series in Hordeum vulgare — a fact which suggests great antiquity for this block of genes.     

Spectrum of chloroperoxidase compound I

When compound I of chloroperoxidase is formed from the native enzyme the absorption peak in the Soret region diminishes in intensity, and shifts to a maximum absorbance at 367 nm. This unusual Soret spectrum decreases in intensity in a linear fashion as the wavelength increases. The first visible spectrum of chloroperoxidase compound I is reported which has a peak at 689 nm as its most prominent feature.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Ontogenetic Variation in the Diurnal Food and Habitat Associations of an Endemic and an Exotic Fish in Floodplain Ponds: Consequences for Niche Partitioning

In floodplain ponds with low piscivore abundance, both endemic Midgley's gudgeons, Hypseleotris sp. 5, and exotic mosquitofish, Gambusia holbrooki, showed significant ontogenetic variation in the use of food and space. Small gudgeons were generally associated with surface and benthic habitats, then restricted their distribution to benthic habitats at a size of approximately 24mm (standard length). The ontogenetic variation in mosquitofish habitat use was less discrete, and could be described as a tendency for larger individuals to be associated with the bottom of the littoral macrophyte beds than with the surface of the macrophyte beds or surface of the limnetic zone. Small gudgeons exhibited high spatial overlap with mosquitofish within the surface habitats of the ponds. All size-class/species comparisons showed significant partitioning of food resources, however, the diets of small gudgeons and mosquitofish were very similar. Therefore, juvenile gudgeons may have to pass through a similar spatial and trophic niche to introduced mosquitofish before recruiting to the adult stage. Possible mechanisms driving the ontogenetic variation in gudgeon and mosquitofish habitat use are discussed. This paper demonstrates that ontogenetic niche shifts at fine spatial scales can affect our interpretation of interactions between native and introduced fishes.