Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

Cyst hatching in Anostraca accelerated by retinoic acid,amplified by Calcium Ionophore A23187, and inhibited by Calcium-channel blockers

Cyst hatching, under standardized conditions, of the Anostracan species Thamnocephalus platyurus and Streptocephalus dichotomus was significantly accelerated but not increased by applying the morphogen retinoic acid (RA). Cyst hatching was enhanced but not accelerated by artificially increasing the inflow of Ca²⁺ to the embryonic cells, using Calcium Ionophore A 23 187. Cyst hatching was accelerated and amplified, to a level in excess of the summed effects of each treatment, by a combined application of RA and ionophore. It was inhibited almost quantitatively by the Calcium-channel blockers Nifedipin and Verapamil. The significance of these findings is discussed.     

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.     

Enterococcal and streptococcal species isolated from faeces of calves, young cattle and dairy cows.

Faeces from non-ruminating calves were found to contain several species of enterococci: Enterococcus avium, Ent. cecorum, Ent. durans, Ent. faecalis, Ent. faecium and Ent. hirae. Enterococcus faecalis was the most frequent. Few of these animals carried streptococci. Streptococcus bovis largely predominated in ruminating calves, young cattle and dairy cows. Other streptococci as well as enterococci were infrequent in dairy cows, but a variety of other streptococci and enterococci were found in the faeces of young ruminating animals.     

Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites.

The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH.     

The effect of feeding on plasma immunoreactive ACTH and beta-endorphin levels in newborn infant.

In order to clarify whether an interaction between endogenous opioids and feeding occurs at birth, we studied Beta-endorphin (beta-EP) and ACTH plasma levels in response to a feed of 10% glucose, or formula, in 120 healthy full-term infants. Neither postprandial beta-EP nor ACTH increases were found at the 24th hour or on the fourth day of life. beta-EP physiology in newborn infants seems to be different from adults.     

Epilithic diatom community response to years of P04 fertilization: Kuparuk River,Alaska (68 N Lat.)

An arctic river was fertilized continuously through the ice-free season with phosphoric acid beginning in 1983. The epilithic diatom community increased in biomass in the first two years in response to the added limiting nutrient (Peterson et al., 1983). The diatom community switched from one dominated by Hannea arcus to one dominated by species of Achnanthes and Cymbella. The immediate responses to the P-addition were decreases in both the Shannon diversity and evenness indices. By the second year, the community diversity increased downriver reaching maximal species richness (110–127 spp). In 1985–1987, the epilithic algal biomass decreased an order of magnitude with both whole-river PO₄ (1985, 1987) and PO₄ + NH₄ addition (1986). In the 5th summer of fertilization, the reduction in biomass was clearly caused by a numerical increase of grazing, refugia-building chironomids (Orthocladiinae, primarily) (Gibeau, 1991; Gibeau, Miller, Hershey, in prep.). We assume the algal biomass reduction in the 3rd and 4th years was similarly caused by grazers with a two year time lag in the numerical response of these monovoltine species. The evenness of the community increased in 1986 as if it might have been grazed; however the number of immigrants was reduced. The community became dominated by Eunotia, Cymbella and Achnanthes, species either fast growing or more prostrate, as the erect species of Hannea Diatoma, and Fragillaria declined. A detrended correspondence analysis of the temporal and spatial diatom samples in species space (186 spp.) showed that the largest variation in the community was between years and less variation was associated with river fertilization. Samples from bioassay tubes run by Peterson et al. (1983) in the Kuparuk River showed P and N + P limitation as found in the river in 1983–84. Like the river samples, the largest change in the diatom community occurred between 15 and 25 day samples, more than that induced by fertilization. Diatoms sampled from all treatments taken at day 25 were more similar to one another than those sampled at day 15. Diatoms colonizing glass slides used in the bioassay tubes were dominated by Achnanthes linearis and Cymbella minuta. Of the 84 species found in bioassays, 26 species were present in all river samples for 4 years. Differences in the communities discriminated by multivariate methods were cause by changes in rare species and abundance patterns of common species.     

Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase.

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.     

O-glycosylation of intact and truncated ribophorins in brefeldin A- treated cells: newly synthesized intact ribophorins are only transiently accessible to the relocated glycosyltransferases

Ribophorins I and II are type I transmembrane glycoproteins of the ER that are segregated to the rough domains of this organelle. Both ribophorins appear to be part of the translocation apparatus for nascent polypeptides that is associated with membrane-bound ribosomes and participate in the formation of a proteinaceous network within the ER membrane that also includes other components of the translocation apparatus. The ribophorins are both highly stable proteins that lack O-linked sugars but each contains one high mannose N-linked oligosaccharide that remains endo H sensitive throughout their lifetimes. We have previously shown (Tsao, Y. S., N. E. Ivessa, M. Adesnik, D. D. Sabatini, and G. Kreibich. 1992. J. Cell Biol. 116:57-67) that a COOH-terminally truncated variant of ribophorin I that contains only the first 332 amino acids of the luminal domain (RI332), when synthesized in permanent transformants of HeLa cells, undergoes a rapid degradation with biphasic kinetics in the ER itself and in a second, as yet unidentified nonlysosomal pre-Golgi compartment. We now show that in cells treated with brefeldin A (BFA) RI332 molecules undergo rapid O-glycosylation in a multistep process that involves the sequential addition of N-acetylgalactosamine, galactose, and terminal sialic acid residues. Addition of O-linked sugars affected all newly synthesized RI332 molecules and was completed soon after synthesis with a half time of about 10 min. In the same cells, intact ribophorins I and II also underwent O-linked glycosylation in the presence of BFA, but these molecules were modified only during a short time period immediately after their synthesis was completed, and the modification affected only a fraction of the newly synthesized polypeptides. More important, these molecules synthesized before the addition of BFA were not modified by O-glycosylation. The same is true for ribophorin I when overexpressed in HeLa cells although it is significantly less stable than the native polypeptide in control cells. We, therefore, conclude that soon after their synthesis, ribophorins lose their susceptibility to the relocated Golgi enzymes that effect the O-glycosylation, most likely as a consequence of a conformational change in the ribophorins that occurs during their maturation, although it cannot be excluded that rapid integration of these molecules into a supramolecular complex in the ER membrane leads to their inaccessibility to these enzymes.