Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii

Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii . The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii .     

A modified mallory-cason staining procedure for large cryosections.

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

The isolation and characterization of gibberellin-deficient mutants in tomato

Summary In tomato, nine independent EMS-induced mutants representing recessive mutations at three different loci (gib-1, gib-2, and gib-3) were isolated. Six of these have an almost absolute gibberellin requirement for seed germination and elongation growth. In addition, the leaves are darker green, smaller, and changed in structure as compared to wild type. The three other mutants, which germinate without GA, are allelic to specific, nongerminating mutants and have less severe mutant characteristics. The respective loci are situated on three different chromosomes. The genes identified by these mutants control steps in gibberellin biosynthesis, as endogenous gibberellins are strongly reduced.     

Influence of dietary fish on eicosanoid metabolism in man

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n-3) and 22:6(n-3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produce HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB2 and TxB3, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyclo-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB2 in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB2 formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGI3, as judged from the appearance of 2,3-dinor-delta 17-6-keto-PGF1 alpha in urine. The amount of the major metabolite of PGI2, 2,3-dinor-6-keto-PGF1 alpha was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.     

Long-term survival of and plasmid stability inPseudomonas andKlebsiella species and appearance of nonculturable cells in agricultural drainage water

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with³²P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.     

Hyphal and mycelial interactions betweenAgaricus bisporus andScytalidium thermophilum on agar media

The interaction betweenAgaricus bisporus andScytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy.A. bisporus combatively replacesS. thermophilum in culture on agar media. The antagonistic effect ofA. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration ofS. thermophilum protoplasm, resulting in a total loss of viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 mm·d^–1 following contact withS. thermophilum mycelium.     

d-Ibuprofen in ocular inflammation induced by paracentesis of the rabbit eye

The anti-inflammatory compound d-ibuprofen has been investigated for anti-inflammatory and cyclooxygenase inhibitory activity in ocular inflammation induced by paracentesis of eyes of living rabbits. d-Ibuprofen is the dextro-rotary isomer of ibuprofen, a potent non-steroidal anti-inflammatory agent. Eyes pretreated topically with d-ibuprofen 0.8% showed a significant inhibition of aqueous protein (73.0%) and prostaglandin E2 (96.4%) increase after paracentesis as compared to paracentesized untreated fellow eyes and control eyes. In aqueous humor no significant correlation between the increase in prostaglandin E2 and protein could be established after paracentesis. These results indicate that d-ibuprofen could be a useful ocular anti-inflammatory agent as cyclooxygenase inhibitor.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.