Stereochemical aspects of the conversion of cyclopeptine into dehydrocyclopeptine by cyclopeptine dehydrogenase from Penicillium cyclopium

Cyclopeptine dehydrogenase, an enzyme from Penicillium cyclopium, catalyses the reversible transformation of the benzodiazepine alkaloids cyclopeptine and dehydrocyclopeptine. By the dehydrogenation of cyclopeptine two hydrogen atoms are removed from the positions 3 and 10. It was demonstrated that, from the two optical isomers of cyclopeptine, only the naturally occurring 3S-compound was used as substrate by cyclopeptine dehydrogenase. To test the stereospecificity of the enzyme with respect to the second hydrogen which is eliminated from C-10 a mixture of cyclopeptine-3S-[10R-³H₁] and cyclopeptine-3R-10S-³H₁] was prepared. The 3S-isomer was transformed by the enzyme into radioactively labelled dehydrocyclopeptine. This demonstrated that cyclopeptine dehydrogenase removes the 10-proS hydrogen atom from the cyclopeptine molecule. Because the formed dehydrocyclopeptine has the trans-configuration it is probable that a synperiplanar elimination takes place. The hydride ion removed from cyclopeptine is transferred to the 4-proR-position of NAD⁺. Cyclopeptine dehydrogenase thus belongs to the A-specific dehydrogenases.     

Dehydrocyclopeptine epoxidase from Penicillium cyclopium

Dehydrocyclopeptine epoxidase (DE) activity was determined in cell free preparations of Penicillium cyclopium. The enzyme transforms dehydrocyclopeptine into cyclopenin by a mixed function oxygenation. It needs molecular oxygen and uses NAD(P)H, ascorbate or d,l-6-methyl-5,6,7,8-tetrahydropteridine as cosubstrates. DE is inhibited by CN^?, SCN^?, 1,10-phenanthroline, EDTA, 2,2′-bipyridine, sodium diethyldithiocarbamate, dicoumarol, p-chloromercuribenzoate and ions of different heavy metals, but not by CO and the lead salt of diethyldithiocarbamate. These properties indicate a specific importance of Fe²⁺-ions, SH-groups and flavins. DE activity is increased by Fe²⁺ and FAD. The enzyme may be therefore a Fe²⁺ activated FAD containing flavoprotein. DE was enriched 268-fold by (NH₄)₂SO₄ precipitation and chromatography on Sephadex G-200. Its MW estimated by Sephadex chromatography, exceeds 480 000.     

Enzymatic synthesis of cyclopeptine intermediates in Penicillium cyclopium

Cyclopeptine, a benzodiazepine alkaloid of Penicillium cyclopium, is formed from anthranilic acid, L-phenylalanine and the methyl group of L-methionine by cyclopeptine synthetase. The following partial activities of this enzyme system were determined in vitro: anthranilic acid and L-phenylalanine adenylyltransferase activity, binding of anthranilic acid and L-phenylalanine as thioesters to proteins, formation of thioester-bound N-methyl-L-phenylalanine and N-methyl-L-phenylalanylanthranilic acid. The obtained results indicate that cyclopeptine is formed via enzyme-bound intermediates by the thiotemplate mechanism of peptide biosynthesis.     

Cyclopenin m-hydroxylase—an enzyme of alkaloid metabolism in Penicillium cyclopium

Cyclopenin m-hydroxylase transforms cyclopenin, one of the two major alkaloids of Penicillium cyclopium, into cyclopenol. The enzyme belongs to the group of mixed function oxygenases. It needs molecular oxygen and a hydrogen donor (NAD(P)H, ascorbic acid, tetrahydropteridine) as cosubstrates, and it is inhibited by CN^? and SCN^? but not by CO. Inhibition by dicoumarol indicates that it may be a flavoprotein. With the exception of the alkaloid viridicatin, all tested compounds structurally related to cyclopenin are hydroxylated. The hydroxylase activity is measurable in the cultures at the end of the growth phase, i.e. during the period of alkaloid metabolism.     

In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa

Of the six carotenoids identified in the cyanobacterium Aphanocapsa, β-carotene, zeaxanthin, echinenone and myxoxanthophyll are the major pigments, whilst β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene are present only in trace amounts. With the exception of zeaxanthin, the other xanthophylls could be formed in vitro from [¹⁴C]phytoene in high yields, especially β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene. In a time course experiment of xanthopyll biosynthesis the flow of radioactivity from [¹⁴C]phytoene was followed through the pools of phytofluene, lycopene, and β-carotene. The reaction sequence from phytoene to xanthophylls is sensitive in vitro to both difunone, an inhibitor of carotene desaturation, and CPTA, an inhibitor of cyclization.     

Channeling of exogenous phenylalanine to the sites of storage and the sites of alkaloid and protein biosynthesis in Penicillium cyclopium

Externally applied l-phenylalanine rapidly equilibrates with the cytosolic pool(s) in hyphae of emerged cultures of Penicillium cyclopium. If not incorporated into protein it is accumulated in the so called expandable pool, which is presumably localized in the vacuolar compartment. At high concentrations of exogenous l-phenylalanine practically all of the amino acid needed for protein synthesis comes from the extracellular source, contrary to alkaloid synthesis which under all conditions recruits more than 90% of the required l-phenylalanine from intracellular sources. Two pathways of alkaloid labelling can be distinguished, by which externally applied l-phenylalanine reaches the sites of alkaloid synthesis: (1) a direct way from cytosolic pool (primary labelling) and (2) an indirect way via the expandable pool (secondary labelling).     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Piperazine derivatives inhibit PrP/PrP propagation in vitro and in vivo

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc^C into a disease-associated isoform PrP^Sc. PrP^Sc accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP^Sc species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP^Sc formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge).     

13C-NMR studies on griseofulvin biosynthesis and acetate metabolism in Penicillium patulum

Incorporations of singly and doubly-labelled acetate-[¹³C] into griseofulvin by a mutant strain of Penicillium patulum confirm its origin from simple folding of a single heptaketide chain. An acetate ‘starter’ effect is observed in the ¹³C-NMR spectra of griseofulvin enriched from acetate-[¹³C], and analysis of the ¹³C—¹³C spin—spin couplings observed indicate a rapid metabolic turnover of added acetate. Methyl, but not carboxyl, of acetate is efficiently metabolised into the C₁ pool.     

Activity of disconnected corpora allata in Locusta migratoria: Juvenile hormone biosynthesis in vitro and physiological effects in vivo

Severance of nervi corporis allati I (NCA I) in day-1 adult female Locusta migratoria resulted in a significant decrease and a loss of the characteristic pattern of juvenile hormone biosynthesis by the corpora allata as determined by radiochemical assay. This decrease in the rate of juvenile hormone biosynthesis was not reflected in basal oöcyte growth. The lengths of the oöcytes were the same in NCA-transectioned and in the sham-operated females. The effect of severance of both NCA I and NCA II on juvenile hormone biosynthesis and ovarian maturation was similar to the effect of NCA I severance only.Rate of juvenile hormone biosynthesis by corpora allata of fourth-instar larvae exhibited a maximum of activity in the middle of the stadium. The severance of NCA I early in the stadium resulted in a very low rate of juvenile hormone biosynthesis and a disappearance of this peak. In NCA I-transectioned larvae, the duration of the stadium was significantly increased although larvae moulted into normal fifth instar.     

Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.     

An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo

Calpain-1 and -2 are Ca^2 +-activated intracellular cysteine proteases that regulate a wide range of cellular functions through the cleavage of their protein substrates. Unlike degradative proteases, calpains make limited, transformative cleavages, typically in accessible sequences linking discrete subdomains, to irreversibly alter substrate functions. The biological roles of calpain and their interplay with calcium signaling are of significant biomedical interest as biomarkers and potential therapeutic targets in a growing number of diseases including Alzheimer's, cancer and fibrosis. Unfortunately, many of the colorimetric and fluorimetric assays that have been developed to study calpain activity suffer from low sensitivity and/or poor calpain specificity. To address the need for a highly sensitive and calpain-specific substrate suitable for in vitro and in vivo calpain activity analysis, we have developed a protein FRET probe. We inserted the optimized calpain cleavage sequence PLFAAR between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) and modulated its flanking sequences for optimal calpain cleavage. We demonstrate greater sensitivity and calpain-specificity of an optimal 16-residue PLFAAR-based FRET substrate compared to a standard α-spectrin-based probe. The 16-residue PLFAAR protein FRET substrate is not significantly cleaved by trypsin, chymotrypsin, cathepsin-L or caspase-3, and is highly sensitive to both calpain-1 and -2. After transfection of the substrate gene into breast cancer cells the PLFAAR protein FRET product was cut in lysed wild-type cells but not in those with a calpain knock-out phenotype. Blockage of substrate cleavage in the lysates by endogenous and exogenous calpastatin was observed, and was overcome by adding extra calpain.     

The diversion of dimethylailylpyrophosphate from polyisoprenoid to cyclopiazonic acid biosynthesis in Penicillium cyclopium westling

During the production of α-cyclopiazonic acid (αCA) by Penicillium cyclopium, dimethylallyltransferase (EC. 2.5.1.1.) T, isopentenyl pyrophosphate isomerase (EC. 5.3.3.2) I, and a prenyl-aryltransferase, S, which produces β-cyclopiazonic acid (βCA) are all induced at the same time. This last enzyme appears maximally before the highest rate of α- or βCA production. Both transferases are not utilized to their maximum capacity, and the production of their end products seems to bear no relationship to their concentrations. Other controls therefore must play an important role in the utilization of their common substrate dimethylallylpyrophosphate (DMAPP). There are two possible control systems: (a), a direct competition by S and T for DMAPP; and (b), control by compartmentation. The first possiblility is the more likely, in view of some of the controls that could apply to the deflection. The three enzymes were separated so that possible controls on the deflection of DMAPP from polyisoprenoids could be studied. They all possessed a subunit structure and exhibited maximum molecular weithts (in the absence of divalent cations and presence of a thiol reductant) of 96 000 (S) and 64 000 (I and T) daltons. Mg²⁺ caused a diminution in size to 75 000 (S) and 50 000 (I and T) daltons. Mg²⁺ had the same effect on I and T but caused major disruptive changes to S. These effects were reversible by addition of EDTA. S was quite specific for DMAPP and cycloacetoacetyl-l-tryptophan (cAATrp) and exhibited Michaelis constants as follows; K_m^cAATrp, 6.0μM and K_m^DMAPP 2.0 μM. It had no obvious requirement for a divalent cation and had an isoelectric point of 5.3. I had a K_m of 6.7 μM and an isoelectric point of 4.5, and either Mg²⁺ or Mn²⁺ was essential. The Michaelis constants for T could not be given but its isoelectric point was 5.1. The enzyme carried out the two reactions normally associated with it (i.e., two additions of IPP to produce farnesyl pyrophosphate) and required Mg²⁺ to do so. The pH optima for S, I, and T were 6.5–7.5, 6.0, and 8.0 respectively. The early and major controlling factor was the appearance of the cosubstrate of S, cAATrp. Other factors were: (a), the appearance of αCA which inhibited T more effectively than S; (b), the removal of free Mn²⁺ and Mg²⁺, both essential for I and T but not for S, possibly brought about by chelation with cAATrp, α- and βCA; (c), the observed low pH of 6.0 when the activity of S was unaltered, I was at its highest, and T exhibited 50% of its maximum; and (d), an activation of I by low physiological levels of βCA and cAATrp which would enhance the rate of appearance of DMAPP to react with an existing pool of cAATrp.     

Inhibition of c-Jun N-terminal kinase increases apoE expression in vitro and in vivo

Apolipoprotein E (apoE), a ligand for the low-density lipoprotein receptor family, has been implicated in modulating glial inflammatory responses and the risk of neurodegeneration associated with Alzheimer’s disease. Glial cells activated by lipopolysaccharide (LPS) have decreased apoE levels and we recently showed that apoE itself can modulate the inflammatory response by reducing c-Jun N-terminal kinase (JNK) activation. Reduced JNK phosphorylation is vital to overcome the LPS-induced decrease in apoE expression, suggesting that JNK inhibition may be an effective way to increase apoE protein and protract its anti-inflammatory properties. This study investigates the impact of JNK inhibition on apoE production using two JNK inhibitors. Our work in primary glia and in vivo in mice injected with JNK inhibitor demonstrates that inhibition of JNK may be an effective way to increase apoE expression.     

In vitro and in vivo digestion of octenyl succinic starch

This study aimed to understand effects of octenyl succinic anhydride (OSA) modification of normal corn (NCS) and high-amylose corn (HA7) starch on their enzymatic hydrolysis rates. After modification with 3% and 10% OSA, resistant starch (RS) contents of the cooked OS-NCS increased from 0.8% of the control starch to 6.8% and 13.2% (Englyst Method), respectively, whereas that of the cooked OS-HA7 decreased from 24.1% to 23.7% and 20.9%, respectively. When the cooked NCS, HA7 and OS (10%)-HA7 were used to prepare diets for rats at 55% (w/w) starch, RS contents of the diets were 1.1%, 13.2% and 14.6%, respectively. After feeding to the rats, 20.2–31.1% of the starch in the OS (10%)-HA7-diet was not utilized in vivo and was found in rat feces, which was substantially larger than that of the HA7-diet (≤4.9%) and NCS-diet (≤0.2%). The body weights of the rats, however, remained similar between different groups.     

Temperature dependence of absorption and fluorescence spectra of bacteriochlorophylls in vivo and in vitro

The “short wave” far-red absorption bands (795–825 nm) of bacteriochlorophyll in photosynthetic red bacteria are sharpened but not shifted upon cooling, the “long wave” far-red bands (840–890 nm) are sharpened less but shifted appreciably towards longer wavelengths. The fluorescence bands are shifted about as much as the corresponding “long wave” absorption bands. Warming results in changes in the opposite direction. The temperature effects are reversible.