A new method for the production of fine plant cell suspension cultures

Fine, almost single cell, suspensions were produced from both existing suspension cultures containing large cell clumps and from chopped callus pieces by immobilizing the cells in 4–5 mm diameter calcium alginate beads. The immobilized cells continued to divide inside the beads and at the bead surface, and after 2–3 weeks' culture, fine cell suspensions were formed as a result of loss of the surface cells into the medium. After removal of the cell suspensions by filtration, subsequent culture of the beads in fresh medium resulted in the further production of homogeneous cell suspensions after 1–2 weeks. In this way an almost continuous supply of fine cell suspensions could be obtained from cultures containing large clumps of cells. The cells produced by this method remained in this state for at least one culture period, although in some instances repeated subculture resulted in an increase in the size of cell groups. The technique has been successfully applied to the production of fine cell suspensions ofCatharanthus roseus, Nicotiana tabacum andDaucus carota.     

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.     

Insertion of a MalE β-Galactosidase Fusion Protein into the Envelope of Escherichia coli Disrupts Biogenesis of Outer Membrane Proteins and Processing of Inner Membrane Proteins

The synthesis of a membrane-bound MalE β-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.     

Selective high-performance liquid chromatographic assays for hydralazine and its metabolites in plasma of man

Selective high-performance liquid chromatographic assays for hydralazine (I), hydralazine pyruvic acid hydrazone (II) and the acetylation metabolites, namely s-triazolo[3,4-a]-phthalazine (V) and 3-hydroxymethyl (VI) and 3-methyl-s-triazolo[3,4-a]phthalazine (VII) in human plasma were developed. Utilizing the fluorescence of these compounds or their derivatives the limits of detection could be extended down to 5 nmole/l (1 ng/ml) for I, 1 nmole/l (0.2 ng/ml) for II and 0.5 nmole/l (0.1 ng/ml) for V–VII. The intra-assay coefficients of variation for the assays ranged from 2 to 7% over the concentration range 5.0 to 0.05 μmole/l and the inter-assay variability in the slope of the standard curves ranged from 4 to 8%. An improved method for measuring the sum of I plus all its hydrazones (apparent I) was also developed. On addition of I to fresh plasma at 37°, half the added I was converted to II within 15 min and there was no detectable level of I, 2 h after the addition. The plasma level—time course of I, and its metabolites in a healthy volunteer (slow acetylator) following separate oral and intravenous administrations of I indicated that I contributed only a small fraction (4.3 and 4.7% respectively) to the area under the plasma level—time curve of apparent hydralazine.     

Transformation of Saccharomyces cerevisiae with yeast mitochondrial DNA linked to two micron circular yeast plasmid

Mitochondrial DNA from a petite mutant of yeast carrying an oligomycin resistance determinant has been ligated in vitro to 2 μm yeast plasmid DNA. The recombinant DNA so produced has been used to transform an oligomycin sensitive strain of Saccharomyces cerevisiae to oligomycin resistance at a frequency approaching 50 times the spontaneous mutation rate to oligomycin resistance. The majority of transformants showed genetic properties suggesting that recombination between the transforming DNA and the resident mtDNA has occurred. The properties of a subclass of oligomycin resistance transformants suggested that in these cells the transforming DNA has not become stably integrated into the mitochondrial genome of the recipient cell.     

The terminal structures of feather keratin mRNA

Terminal labeling of embryonic feather keratin mRNA with [³H]KBH₄ followed by digestion with ribonuclease T₁ and T₂, alkaline phosphatase, snake venom phosphodiesterase, and nucleotide pyrophosphatase and analysis of the products by high voltage paper electrophoresis, indicated the presence of the sequence m⁷G(5)ppp(5)N at the 5-end of the mRNA. Ribonuclease T₁ and A digests of the terminally labeled, and also of unlabeled mRNA followed by fractionation on denaturing polyacrylamide gels indicated the presence of polyadenylate tracts ranging in size from 45 to 165 nucleotides at the 3-end of the mRNA.     

Halogenated monoterpenes of the red alga Microcladia

The red marine algae Microcladia borealis, M. californica and M. coulteri produce several unusual halogenated monoterpenes including violacene, plocamene-B, plocamene-C, and plocamane-D. The isolation of these terpenes along with a study of their variation in each Microcladia at different locations are described.