Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

Photoperiod control of poplar bark storage protein accumulation

Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.     

Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Intramitochondrial factors controlling hepatic fatty acid oxidation at weaning in the rat

Fatty acid oxidation was studied in isolated liver mitochondria of rats during the suckling-weaning transition. The oxidation rate of oleyl-CoA and palmitoylcarnitine was reduced 2.5-fold in rats weaned on a high-carbohydrate diet compared to suckling rats, when acetyl-CoA produced by beta-oxidation was directed towards ketone-body synthesis. Weaning on a high-fat diet minimized this change. Channeling of acetyl-CoA towards citrate synthesis doubled the oxidation rate of both substrates in HC-weaned rats. Thus, in addition to changes in carnitine palmitoyltransferase I activity, the beta-hydroxymethylglutaryl-CoA synthase pathway is also involved in the decreased fatty acid oxidation at weaning. This was confirmed by measurement of beta-hydroxymethylglutaryl-CoA synthase pathway activity.     

Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide

The expression of two membrane glycoproteins, RL388 antigen and transferrin receptor (TfR), was examined on murine B cells stimulated with lipopolysaccharide (LPS) in vitro. Immunofluorescent staining with monoclonal antibodies and flow cytofluorometric analysis were used to monitor the expression of these markers as a function of the time in culture, the state of membrane Ia antigen expression, the position in cell cycle, and the degree of B-cell differentiation. Freshly explanted splenic B cells expressed low levels of RL388 antigen and TfR. Following LPS stimulation, increased expression of RL388 antigen was detectable by 8 to 12 hr of culture, a time span characterized by increased Ia antigen expression, blast transformation, and G0 to G1 phase transition. The increased expression of TfR was apparent later and correlated with entry into late G1 phase and the onset of S phase. LPS-stimulated cell cultures treated with actinomycin D (G0/G1 block) exhibited increased expression of Ia antigen, but neither RL388 antigen nor TfR, whereas hydroxyurea treatment (G1/S block) allowed expression of all three markers. These results indicate that hyperexpression of RL388 antigen and TfR occurs during G1 phase and that these events are subsequent to Ia antigen hyperexpression. Finally, B cells in late G1 through M phase of the cell cycle simultaneously express high levels of RL388 antigen and TfR. These findings suggest that the expression patterns of RL388 antigen and TfR might be useful parameters for defining compartments of the murine B-cell cycle.     

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.     

Pollen as a chronometer and sediment tracer,Burrinjuck Reservoir,Australia

Pollen analysis is widely used to reconstruct vegetation and land use histories, but can also provide sedimentological information. At Burrinjuck Reservoir, in south-eastern Australia, annual grass pollen peaks are used to distinguish each year's sediment, even when there are no visible laminations. In conjunction with other dating methods, this allows the determination of year by year influxes of all sediment components. Pollen grains in the Burrinjuck sediments are shown to be predominantly waterborne so that they can be used to trace sediment to its source in particular vegetation stands. Pollen concentration and the proportion of damaged pollen might also distinguish sediment eroded from topsoils and that from subsoils. Pollen analysis can thus be used to locate specific erosion events in both time and space.     

Sequence homologies of glucose-dehydrogenases of Bacillus megaterium and Bacillus subtilis

The sequence homologies of the glucose dehydrogenase subunits of B. megaterium and B. subtilis are compared. From the known B. megaterium aminoacid sequence and the base sequence of the cloned B. subtilis structural gene we predict the B. megaterium structural glucose dehydrogenase gene. Assuming the minimal mutational changes to convert one gene into the other 23 transitions, 30 transversions, 1 inversion, 3 insertion-deletions, but no frameshifts are postulated necessary to interconvert the structural genes. The homology of both enzyme subunits of 85% reflects the close evolutionary distance between B. subtilis and B. megaterium.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.