Naturally occurring monoterpenoids in needles of Picea abies (L.) Karst

Summary An analysis was made of the effects of different sampling and extraction techniques on the amounts and pattern of monoterpenoids isolated from needles of Norway spruce. The following isolation and analysis procedure was finally adopted: liquid nitrogen-cooled needles were pulverized by a microdismembrator, extracted with pentane overnight at 2°–3°C and concentrated to a volume not less than 3 ml/g fresh weight on a Vigreux column. The crude extract was injected splitless (with solvent split) onto a cold programmed temperature vaporized (PTV) precolumn of a gas chromatograph and the vaporizable compounds heated to a capillary column. This method was tested for production of artefacts and quantitative extraction and applied to needles of eleven 80-year-old spruce trees.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Effect on aflatoxin production of competition between wild-type and mutant strains of Aspergillus parasiticus

Co-cultivation of a strain of Aspergillus parasiticus, capable of making aflatoxins, with blocked mutant strains, capable of producing none or only a low level of aflatoxins, reduced the net yield of aflatoxins more than that expected based on spore recovery. Yields of aflatoxins were 8-fold less for a norsolorinic acid-producing strain, 14-fold less for an averantin-producing strain, 6-fold less for an averufin-producing strain, and 21-fold less for a versicolorin A-producing strain when co-cultured in equal amounts with a wild-type strain of Aspergillus parasiticus. Even when the wild-type strain was initially present in 100-fold excess, with two of the mutant strains, reduced aflatoxin production was still observed.     

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

A new gene controlling the frequency of cell division per round of DNA replication in Escherichia coli

Summary A novel mutant of Escherichia coli, named cfcA1, was isolated from a temperature-sensitive dnaB42 strain, and found to have the following characteristics. Division arrest and lethality induced by inhibition of DNA replication was reduced and delayed in the cfcA1 dnaB42 strain, as compared with the parental dnaB42 strain. Two types of inhibition of division induced by the addition of nalidixic acid or hydroxyurea were suppressed by the cfcA1 mutation. Under permissive conditions for DNA replication, the colony forming ability of cfcA1 cells was significantly reduced as compared with that of cfc ⁺ cells; conversely the division rate of cfcA1 cells was higher than that of cfc ⁺ cells. The cfcA1 mutation partially restored division arrest induced in the thermosensitive ftsZ84 mutant at the restrictive temperature and suppresed the UV sensitivity of the lon mutation. The mutation was mapped at 79.2 min on the E. coli chromosome. Taking these properties into account, it is hypothesized that the cfcA gene is involved in determining the frequency of cell division per round of DNA replication by interacting with the FtsZ protein which is essential for cell division.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Role of phospholipases in myocardial ischemia: effect of cardioprotective agents on the phospholipases A of heart cytosol and sarcoplasmic reticulum in vitro

Increased breakdown of myocardial phospholipids to fatty acids and lysophosphoglycerides is an early feature of myocardial ischemic injury and many investigators believe that enhanced phospholipase action is an important factor in the process. Several recent reports indicate that inhibitors of phospholipase A, such as mepacrine, chloroquine and chlorpromazine, can prevent heart phosphoglyceride breakdown in vivo. We isolated the phospholipases A from rat heart cytosol and sarcoplasmic reticulum and examined the effects of various cardioprotective substances on their activity. Most of the cardioprotective agents studied inhibited the heart phospholipases in vitro, providing further evidence that phospholipid degradation in ischemic myocardial injury may be modulated by pharmacologic agents.