The role of l-phenylalanine in the production of isobutene by Rhodotorula minuta

The role of l-phenylalanine and its synergistic effect on the production of isobutene were investigated with both the living cells and a cell-free system of Rhodotorula minuta IFO 1102. Many aromatic carboxylic acids also had the same effect on the production of isobutene as l-phenylalanine. Cycloheximide, an inhibitor of protein synthesis, inhibited the synergistic effect of l-phenylalanine on the production of isobutene. Furthermore, the cell-free extract prepared from cells cultivated in the medium which contained l-phenylalanine had isobutene-forming activity. These results confirm that l-phenylalanine is an inducer of a tentative “isobutene-forming enzyme” in Rhodotorula minuta.     

High-level production of soluble adenine nucleotide translocator from Schistosoma japonicum in E. coli cell-free system

Adenine nucleotide translocator is an important member of the mitochondrial carrier family, which is involved in the transportation of various metabolites. In the present work, the Escherichia coli cell-free system was chosen as an alternative way to express this highly toxic membrane protein. The expression level of insoluble sjANT from Schistosoma japonicum in E. coli cell-free system could attain 472 μg/mL, about 25.2 times improvement over the previous report. The obtained insoluble sjANT can be resolubilized with different detergents. Among them, Digitonin could effectively solubilize approximate 38% of the target membrane protein. Moreover, sjANT can be further expressed in the hydrophobic E. coli cell-free system as a soluble form with presence of different detergents. The results suggested that Digitonin and Brij 58 were two ideal candidates to support high expression of soluble sjANT, and the highest soluble expression level (182 μg/mL) was achieved with the supplementation of 0.4% (v/v) Digitonin in the cell-free system. The present work has provided a rapid and efficient procedure to express the complex and highly toxic membrane protein in the cell-free system, and will be beneficial to construct a novel drug-discovery model to screen the sjANT-based inhibitors for schistosomiasis treatment.     

Preparation of Efficient Excision Repair Competent Cell-Free Extracts from C. reinhardtii Cells

Chlamydomonas reinhardtii is a prospective model system for understanding molecular mechanisms associated with DNA repair in plants and algae. To explore this possibility, we have developed an in vitro repair system from C. reinhardtii cell-free extracts that can efficiently repair UVC damage (Thymine-dimers) in the DNA. We observed that excision repair (ER) synthesis based nucleotide incorporation, specifically in UVC damaged supercoiled (SC) DNA, was followed by ligation of nicks. Photoreactivation efficiently competed out the ER in the presence of light. In addition, repair efficiency in cell-free extracts from ER deficient strains was several fold lower than that of wild-type cell extract. Interestingly, the inhibitor profile of repair DNA polymerase involved in C. reinhardtii in vitro ER system was akin to animal rather than plant DNA polymerase. The methodology to prepare repair competent cell-free extracts described in the current study can aid further molecular characterization of ER pathway in C. reinhardtii.     

Certain properties of isoniazid inhibition of mycolic acid synthesis in cell-free systems of M. aurum and M. avium

In Mycobacterium tuberculosis isoniazid (INH)-susceptibility and the presence of a thermolabile catalase-peroxidase (T-catalase) are nearly always associated. It is shown in this study that an INH-susceptible strain of M. aurum had a T-catalase activity while its resistant mutants did not, but an in vitro susceptible strain of M. avium had a strong catalase activity without any detectable peroxidase properties. Synthesis of mycolic acids is a genus-specific target for INH and there is an excellent parallelism between INH-susceptibility of intact cells and that of a cell-free system synthesizing mycolic acids. We investigated whether the INH-inhibition of mycolic acid cell-free synthesis was dependent on a T-catalase activity in M. aurum and M. avium: no catalase activity was detectable in any of the cell-free systems tested, and addition of T-catalase from susceptible M. aurum strain to an INH-resistant system did not render it sensitive. So INH can inhibit mycolic acid synthesis independently of the presence of a T-catalase. An INH-susceptible cell-free system prepared from INH-treated (at the MIC) cells was progressively and irreversibly inhibited, while incubation of the same susceptible system in the presence of INH did not result in a significant irreversible inhibition. The possible participation of T-catalase in the irreversible effect of INH is discussed.     

Oligomannoside biosynthesis in Mycobacterium smegmatis

A cell-free particulate enzyme system of Mycobacterium smegmatis ATCC 607 was shown to catalyze the incorporation of labeled mannose from GDP-[¹⁴C]mannose into endogenous acceptors to form a series of labeled neutral oligomannosides. These oligomannosides were devoid of amino sugar. The major oligomannoside product was characterized to be a trimannoside, O-α-d-mannopyranosyl-(1 → 2)-O-α-d-mannopyranosyl(1 → 2)-d-mannose and represented 46% of the total labeled oligomannoside product. The higher oligomannosides were shown to have either/or both α(1 → 2) and α(1 → 6) glycosidic linkages. A series of unlabeled endogenous oligosaccharides was isolated from the 105,000g supernatant fractions of the cell-free extracts of M. smegmatis and found to be chromatographically similar to the labeled oligomannosides synthesized by the cell-free system. The nature of the endogenous acceptor was not determined.     

Efficient production of soluble human beta-defensin-3–4 fusion proteins in Escherichia coli cell-free system

Human β-defensin-3 and 4 (HBD-3–4) are two low molecular weight cationic peptides with three conserved cysteine disulfide bonds, and exhibit a broad range of antimicrobial activity and do not acquire any microbial resistance. In order to produce these uneasily detectable, degradable and toxic polypeptide efficiently, an alternative approach based on the Escherichia coli cell-free biosynthesis system was proposed. The polypeptide of interest is synthesized as a fusion protein linked to trxA or green fluorescent protein (GFP) through a cleavable spacer. With batch mode operation, significant amount of hBD3–4 fused with trxA or GFP can be expressed in this cell-free system, and the product is soluble and stable. Furthermore, the GFP moiety provides directly visuable and quantitative monitoring of the polypeptide synthesis. This work will be helpful to rapid and visuable expression of other similar defensins using in vitro cell-free system.     

Baciphelacin: a new eukaryotic translation inhibitor

Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10⁻⁷ M inhibited protein synthesis by 50% in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin.     

Kaurenolide biosynthesis in a cell-free system from Cucurbita maxima seeds

A new product obtained by incubation of [2-¹⁴C ]-mevalonic acid with a cell-free system from Cucurbita maxima endosperm was identified by GC-MS as ent-kaura-6,16-dien-19-oic acid. When this compound was reincubated with the microsomal fraction it was converted to 7β-hydroxykaurenolide and hence to 7β,12α-dihydroxykaurenolide. The dienoic acid was also obtained by incubation of ent-kaurene, ent1-kaurenol, ent-kaurenal and ent-kaurenoic acid, but not ent-7α-hydroxykaurenoic acid, with the microsomal fraction. Thus, in the C. maxima cell-free system, the kaurenolides are formed by a pathway which branches from the GA pathway at ent-kaurenoic acid and proceeds via the dienoic acid.     

Cell-free synthesis of Chironomus thummi (Diptera) globins

RNA isolated from Chironomus thummi (Diptera) larvae directs the incorporation of amino acid into newly synthesized products in a cell-free translation system prepared from wheat germ. A fraction of the total cell-free product was specifically immunoprecipitable with antibody against total C. thummi hemoglobin. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the immunoreactive material revealed the cell-free product to have an apparent molecular mass approximately 3000 daltons greater than secreted C. thummi globin purified from hemolymph. In contrast, analysis of the immunoreactive material by polyacrylamide gel electrophoresis under nondenaturing conditions indicated several chemically distinct globins to be present in the cell-free immunoreactive products. These results provide evidence suggesting the possible existence of a preglobin and the data further provide the initial foundation required for elucidating the regulatory mechanisms that control the developmental stage-specific expression of the globin genes in C. thummi.     

Aldrin epoxidation by plant root extracts

Aldrin epoxidase activity of the cell-free pea and bean root preparations was located in the particulate fraction. High speed centrifugation at 250 000 g for 2 hr resulted in a pellet with almost all the activity of the crude cell-free preparations. While the epoxidase was stimulated by NADPH generating system in these cell-free root preparations, that in high speed centrifugation pellets was not. Aldrin epoxidase activity of the dwarf bean root homogenates was increased by addition of p-aminobenzoic acid (10^?4 M). This increase in activity is above that already manifested by Polyclar AT. No activity was detected in the dormant or germinating Alaska peas or dwarf beans until the 5th and 6th day, respectively.     

Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate

Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59 136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine     

Prostaglandin E1 and analogs of prostacyclin influencing superoxide production by the human neutrophil nadph oxidase system

• 1.1. The effects of prostaglandin (PG) E₁, and I₂ analogs (OP-41483 and OP-2507) on the Superoxide generation of human neutrophil NADPH oxidase (EC 1.6.99.6) in both whole-cell and cell-free systems were investigated.
• 2.2. In a whole-cell system, OP-2507 inhibited the Superoxide generation by neutrophils exposed to phorbol myristate acetate concentration-dependently through its superoxide-scavenging action.
• 3.3. The concentration of the drug required for 50% inhibition of the oxidase (IC₅₀) was 21 μM.
• 4.4. In a cell-free system, however, the drug in concentrations of < 100 μM did not inhibit the activation of NADPH oxidase by sodium dodecyl sulfate because of its inactivation by the detergent.
• 5.5. Although PGE₁ and OP-41483 did not inhibit the Superoxide production by stimulated neutrophils in a whole-cell system, they both inhibited the activation of NADPH oxidase in a cell-free system concentration-dependently, with IC₅₀ values of 44 and 170 μM, respectively.
• 6.6. In addition, in the cell-free system, the K_m value for NADPH of the oxidase was unchanged by PGE₁.
• 7.7. The results suggest that the PGI₂ analog, OP-2507, is a possible superoxide-scavenger and that PGE₁ inhibits the NADPH oxidase activation by sodium dodecyl sulfate in a cell-free system concentration-dependently.

     

Construction of arsenic-resistant Thiobacillus ferrooxidans recombinant plasmids and the expression of autotrophic plasmid genes in a heterotrophic cell-free system

Recombinant plasmids between Thiobacillus ferrooxidans and Escherichia coli which contain arsenic and antibiotic resistance genes and unique restriction sites have been constructed. The expression of autotrophic T. ferrooxidans genes in a heterotrophic E. coli cell-free system was demonstrated.     

Relation between nuclear envelope and nuclear lamina in nuclear assemblyin vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Mevalonate-activating enzymes in callus culture cells from Nepeta cataria

The 30000 g supernatants from cell-free extracts of Nepeta cataria leaf tissue and leaf callus tissue have mevalonic acid kinase, mevalonic acid phosphate kinase and mevalonic acid pyrophosphate decarboxylase activities. The callus tissue cell-free extract produced mevalonic acid pyrophosphate and isopentenyl pyrophosphate; however, very little mevalonic acid phosphate was observed. The leaf cell-free extracts incubated with [¹⁴C]-mevalonic acid produced higher amounts of mevalonic acid phosphate. When both the leaf cell-free extract and the callus cell-free extract were incubated with [¹⁴C]-mevalonic acid in the presence of iodoacetamide, the ion exchange column elution profile was cleaner, which was confirmed by PC. Apparently the callus tissue 30000 g supernatant contains mevalonic acid phosphorylating enzymes even though there is no production of the methyl cyclopentane monoterpenes.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Misreading of the ribosomal suppressor SUP46 due to an altered 40 S subunit in yeast

The dominant suppressor, SUP46, in the yeast Saccharomyces cerevisiae acts on a wide range of different types of mutations. The incorporation of phenylalanine and the misincorporation of leucine in a cell-free system programmed with poly(U) indicated that the ribosomes from a SUP46 strain produce abnormally high rates of translation errors. Furthermore, the cell-free translation system was used to demonstrate that the SUP46 defect resides in the 40 S ribosomal subunit. The growth of SUP46 strains was shown to be unusually sensitive to paromomycin, an aminoglycoside antibiotic that is known to induce translation errors. In addition, paromomycin stimulated mistranslation with SUP46 ribosomes to a greater extent than with normal ribosomes. These results indicate that SUP46 suppression is caused by increased translation errors as a result of the mutationally altered 40 S ribosomal subunit. Paromomycin appears to produce translation errors in SUP46 strains at rates that are too high for cellular growth.     

A membrane-bound isobutene-forming enzyme from Rhodotorula minuta

Abstract An Arrhenius plot for the formation of isobutene by cell extracts of Rhodotorula minuta IFO 1102 gave a discontinuous line and isobutene-forming activity was found to be present in the microsomal fraction of the extracts. These results indicate that the isobutene-forming enzyme is membrane-bound.
The formation of isobutene by the microsomes was inhibited by some redox reagents and completely eliminated by the presence of carbon monoxide. These findings suggested that a cytochrome P-450 participated in the reaction. In fact, a cytochrome P-450 and cytochrome P-450 reductase were detected in the microsomes obtained from cells that contained the isobutene-forming activity.     

Comparison of ent-kaurene and ent-isokaurene synthesis in cell-free systems from etiolated shoots of normal and dwarf-5 maize seedlings

An active cell-free system, prepared from young etiolated shoots of normal Zea mays seedlings, was shown to biosynthesize the terpenoid hydrocarbons ent-kaur-16-ene, squalene and phytoene from mevalonic acid. The biosynthesis of ent-kaur-16-ene from mevalonic acid was compared using cell-free systems obtained from normal and dwarf-5 seedlings. ent-Kaur-16-ene was the predominant diterpene hydrocarbon synthesized by extracts from the normals; however, ent-kaur-15-ene was the major diterpene hydrocarbon synthesized by the dwarf-5 mutants. ent-Kaur-15-ene and ent-kaur-16-ene were also produced as minor products in the normal and dwarf-5 systems, respectively. The possible significance of the synthesis of the ‘wrong isomer’ (ent-kaur-15-ene) by the mutant is discussed.