Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Cost implications of the British Pacing and Electrophysiology Group's recommendations for pacing.

OBJECTIVE--To compare present pacing practice with the recommendations recently published by the British Pacing and Electrophysiology Group and to assess the increase in annual budget required to implement these recommendations in a regional cardiothoracic unit. DESIGN--Retrospective analysis of pacemaker implantation for 1991 with calculation of the costs required to implement the group''s recommendations based on average 1991 costs of the types of pacing generators and electrode leads used. SETTING--Regional cardiothoracic unit for South West Thames Health Authority. PATIENTS--433 consecutive patients receiving permanent pacemaker generators: 76 (18%) with sinus node disease; 270 (62%) with atrioventricular block; 25 (6%) with both sinus node disease and atrioventricular block; 59 (14%) with chronic atrial fibrillation and atrioventricular block; and 3 (1%) with carotid sinus or malignant vasovagal syndromes. RESULTS--Only 102 (24%) patients received pacemaker generators recommended by the British Pacing and Electrophysiology Group; however, 355 (82%) patients were older than 65 years, and 264 (61%) were aged 75 or over. The cost of hardware for pacing was 462,885 pounds. Using generators as recommended would have cost 810,525 pounds for "optimal" systems (an increase of 75%) and 710,750 pounds for "alternative" systems (an increase of 54%). These increases would have been considerably reduced by limiting the use of sophisticated pacing to younger patients (aged under 75). Further savings could be made by using the least expensive pacing models available. CONCLUSIONS--Implementing these recommendations should reduce morbidity related to bradyarrhythmia but will lead to major increases in pacing costs. Age and patients'' expected activity may be used to select simple pacing systems and thus to contain cost. More research is needed to determine which patient groups will benefit most from complex pacing systems.     

Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy     

A new allele of the duplicated 27kD zein locus of maize generated by homologous recombination.

The 27kD zein storage protein locus in many inbred lines of maize consists of a tandem duplication of 12kb, with an expressed gene in each repeat, termed A and B. A single-copy allele with only the A gene can be generated from this duplication in particular stocks of the maize inbred line A188 by a mitotic process that includes a crossover at the 3' regions of the two genes (1). Here, we characterize a second single-copy allele with only the B gene, found in different stocks of A188. This allele arises from a homologous recombination at the highly conserved 5' regions of the two repeats, and cloning and sequencing of this allele define the crossover region. The A and B genes in the duplicated allele were previously shown to be expressed at different levels; this difference remains unchanged in either recombinant allele. Therefore, the crossover points of these two recombinant alleles define the borders of cis-acting sequences responsible for the differential expression of the two genes.     

Purification, properties and cation activation of galactosyltransferase from lactating-rat mammary Golgi membranes

Galactosyltransferase was purified from Golgi membranes of lactating-rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties. The enzyme occurred in both monomeric (43-46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N-terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro-Pro-Pro-Pro sequence. Kinetic studies demonstrated a high-affinity Mn2+-binding site (1) essential for activity, and a low-affinity Mn2+-binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase. It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.     

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats

Summary The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.     

Tyrosyl-tRNA synthetase acts as an asymmetric dimer in charging tRNA. A rationale for half-of-the-sites activity

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a classical example of an enzyme with half-of-the-sites activity. The enzyme crystallizes as a symmetrical dimer that is composed of identical subunits, each having a complete active site. In solution, however, tyrosyl-tRNA synthetase binds tightly, and activates rapidly, only 1 mol of Tyr/mol of dimer. It has recently been shown that the half-of-the-sites activity results from an inherent asymmetry of the enzyme. Only one subunit catalyzes formation of Tyr-AMP, and interchange of activity between subunits is not detectable over a long time scale. Paradoxically, however, the kinetics of tRNA charging are biphasic with respect to [Tyr], suggesting that both subunits of the dimer are catalytically active. This paradox has now been resolved by kinetic analysis of heterodimeric enzymes containing different mutations in each subunit. Biphasic kinetics with unchanged values of KM for Tyr are maintained when one of the two tRNA-binding domains is removed and also when the affinity of the "inactive" site for Try is reduced by 2-58-fold. The biphasic kinetics do not result from catalysis at both active sites, but instead appear to result from two molecules of Tyr binding sequentially to the same site. A second molecule of Tyr perhaps aids the dissociation of Tyr-tRNA by displacing the tyrosyl moiety from its binding site. A monomer of the enzyme is probably too small to allow both recognition and aminoacylation of a tRNA molecule. This could explain the requirement for the enzyme to function as an asymmetric dimer.     

Asymmetry of tyrosyl-tRNA synthetase in solution

The tyrosyl-tRNA synthetase from Bacillus stearothermophilus crystallizes as a symmetrical dimer with each subunit having a complete active site. The enzyme-substrate complexes, however, are known to be asymmetrical in solution because the enzyme exhibits half-of-the-sites activity by binding tightly only 1 mol of tyrosine or 1 mol of tyrosyl adenylate per mole of dimer. Evidence is now presented that the unligated enzyme is also asymmetrical in solution. Symmetry was investigated by construction of heterodimers containing one full-length subunit and one truncated subunit, allowing the introduction of different mutations into each monomer. Each dimer is active at only one site, but the site used is randomly distributed between the subunits. Each heterodimer thus consists of two equal populations, one activating tyrosine at a full-length subunit and the other at the truncated subunit. No detectable interconversion is found between active and inactive sites over several minutes either in the absence of substrates or when the enzyme is turning over in the steady state. Kinetic evidence implies that wild-type enzyme is inherently asymmetrical even in the absence of substrate.     

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.