Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

Atrial peptide inactivation by rabbit-kidney brush-border membranes

Atriopeptin (AP) 24, containing amino acids Ser103-Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103-Ser104, Cys105-Phe106 and Ser123-Phe124. des-Ser103-AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at Cys105-Phe106 was inactive. Regarding the proteolytic cleavage at Ser123-Phe124, there was an accumulation of the C-terminal tripeptide, Phe-Arg-Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino- and carboxy-terminal protected peptide, acetyl-AP24-amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was Cys105-Phe106. We conclude that the Cys-Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.     

Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.     

The effect of feeding endophyte infected tall fescue seed on reproductive performance in female rats

Female Sprague-Dawley rats (Rattus norvegicus) were randomly assigned to diets containing mixtures of rat chow and tall fescue (Festuca arundinacea) seed with 0, 5, 10, 20 and 40% infection levels of Acremonium coenophialum to assess the effect of the diets on the reproductive potential of rats. Rats fed 40% infected seed had decreased body weight, decreased mean percent body weight of uteri, failed to maintain normal estrous cycles and were unable to become pregnant. Animals fed a diet of 20% infected fescue seed had extended estrous cycles. There were no significant differences among the 0, 5 and 10% dietary treatments.     

Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy.

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 2. Fe4S4 cluster vibrational modes

Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand.     

Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases

In the preceding paper (Ellens et al., 1989), it was shown that liposome fusion rates are substantially enhanced under the same conditions which induce isotropic 31P NMR resonances in multilamellar dispersions of the same lipid. Both of these phenomena occur within the same temperature interval, delta TI, below the L alpha/HII phase transition temperature, TH. TH and delta TI can be extremely sensitive to the lipid composition. The present work shows that 2 mol% of diacylglycerols like those produced by the phosphatidylinositol cycle in vivo can lower TH, delta TI, and the temperature for fast membrane fusion by 15-20 degrees C. N-Monomethylated dioleoylphosphatidylethanolamine is used as a model system. These results show that physiological levels of diacylglycerols can substantially increase the susceptibility of phospholipid membranes to fusion. This suggests that, in addition to their role in protein kinase C activation, diacylglycerols could play a more direct role in the fusion event during stimulus-exocytosis coupling in vivo.     

Sexual receptivity in hamsters: brain nuclear estrogen and cytosolic progestin receptors after single and multiple steroid treatments and during the estrous cycle

This series of experiments investigated the relationship between various treatments consisting of estradiol benzoate (EB) and progesterone (P) on sexual receptivity and on concentrations of nuclear estrogen receptors (NER) and cytosolic progestin receptors (CPR) in the hypothalamus-preoptic area in female hamsters. The injection of 1 microgram EB at 0 and 24 hr resulted in higher levels of receptivity (after 0.25 or 0.5 mg P), NER and CPR compared to those obtained after a single injection of 2 micrograms EB. Animals treated with 5 micrograms EB at 0 and 24 hr displayed greater levels of receptivity (after 0.5 mg P) and had higher NER concentrations than animals given a single injection of 10 micrograms EB. Groups treated with either 1 microgram EB at 0 hr or 0.5 microgram EB at 0 and 24 hr did not differ and showed low levels of receptivity, NER and CPR, NER and CPR were also measured on each day of the estrous cycle. NER levels rose between Days 1 and 2, again between Days 2 and 3, and remained elevated on Day 4. CPR levels increased between Days 2 and 3, and there was no difference between Days 3 and 4. Taken together, these data suggest that receptivity in hamsters after estrogen exposure is correlated with the accumulation and maintenance of relatively high NER levels and on the induction of CPR. This can be accomplished by a single large injection of EB or by smaller split doses.     

Variability in the macrozooplankton community off the Antarctic Peninsula

Summary This paper presents an analysis of zooplankton net sampling surveys carried out during four expeditions to the Antarctic Peninsula region. Cluster analysis documented two to three site groupings for the epipelagic zone and one mesopelagic site cluster below 200 to 300 m depth. Analysis of species dominance, constancy, diversity and evenness indices did not allow clear designation and separation of communities in terms of these parameters.Computation of a rank correlation matrix for each season allowed the characterization of species groups. There were no perfect indicator species in the very strict sense. The main differences in the composition of the zooplankton between the site clusters were due mainly to changes in abundance rather than to presence or absence of particular species. However, the interpretation of the complex species and site groupings led to the conclusion that we can define three distinct communities: an oceanic, a neritic, and a mesopelagic community beneath 200 to 300 m. A so-called transitional cluster represents a mixing zone created by frequent occurrence of species from both the oceanic and neritic community. The location of the described oceanic and neritic community sites seem to be relatively stable with minor latitudinal changes during the seasons, while occurrence and abundance of most species changes with the time of the year. The usefulness of particular species (e.g. Euphausia superba) as indicator species also change during the year     

Home range and habitat use of wolverines Gulo gulo in Yukon, Canada

Home ranges and habitat use are described for three adult female, one adult male, and one subadult male wolverines in the Kluane Game Sanctuary, Yukon. When long distance excursions are not included, home ranges of wolverines in the Kluane Game Sanctuary were between 76 and 269 km² for females and 209 and 269 km² for males. Habitat use of females was similar to habitat availability. Males used subalpine coniferous habitats more frequently than other habitat types during winter. Although individual variation in the use of forest cover types, aspects, slopes and elevations was apparent, seasonal use did not differ from availability for each sex. Within the 1590 km² study area, three adult males and six adult females were present, corresponding to a density of one resident wolverine 177 km^-2.