Proteasome and NF-kappaB inhibiting phaeophytins from the green alga Cladophora fascicularis

Chemical examination of the green alga Cladophora fascicularis resulted in the isolation and characterization of a new porphyrin derivative, porphyrinolactone (1), along with five known phaeophytins 2-6 and fourteen sterols and cycloartanes. The structure of 1 was determined on the basis of spectroscopic analyses and by comparison of its NMR data with those of known phaeophytins. Compounds 1-6 displayed moderate inhibition of tumor necrosis factor alpha (TNF-alpha) induced nuclear factor-kappaB (NF-kappaB) activation, while 2 and 4 displayed potential inhibitory activity toward proteasome chymotripsin-like activation. The primary structure-activity relationship was also discussed.     

Steroid and antibacterial steroidal glycosides from marine green alga Codium iyengarii Borgesen

The ethyl acetate soluble part of methanolic extract of marine green alga Codium iyengarii collected from Karachi coast of Arabian Sea afforded a new steroid ([structure: see text], iyengadione) and two new steroidal glycosides [iyengaroside-A ([structure: see text]) and B ([structure: see text])] along with clerosterol galactoside ([structure: see text]). Their structures were elucidated with the aid of 1D-NMR spectroscopy and reconfirmed through HMBC experiments. The bactericidal activity of [structure: see text] was also explored and found positive response from iyengaroside-A ([structure: see text]) and clerosterol galactoside ([structure: see text]).     

Technetium uptake by the green alga Chlorella Fusca

A transfer factor of 0.16±0.07 (Bq g^–1 fresh algae: Bq ml^–1 solution) was found for the uptake of TcO₄ ^– by the green alga Chlorella fusca.     

The photoreactions of recombinant phytochrome from the cyanobacterium Synechocystis: a low-temperature UV-Vis and FT-IR spectroscopic study

The interconvertible photoreactions of recombinant phytochrome from Synechocystis reconstituted with phycocyanobilin were investigated by light-induced optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be deferred below -100 degrees C for the transformation of red-absorbing form of phytochrome (Pr)-->far-red-absorbing form of phytochrome (Pfr), and no formation of an intermediate similar to the photoproduct of phytochrome A obtained at -140 degrees C (lumi-R) was observed. Two intermediates could be stabilized below -40 degrees C and between -40 and -20 degrees C, and were denoted as meta-Ra and meta-Rc, respectively. Above -20 degrees C Pfr was obtained. In the reverse reaction two intermediates could be stabilized below -60 degrees C (lumi-F) and between -60 and -40 degrees C (meta-F). The FT-IR difference spectra of the late Pr-->Pfr photoreaction show great similarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environment, whereas deviations in the spectra of meta-Ra were observed. A large band around 1700 cm-1 in the difference spectra between the intermediates and Pr which is tentatively assigned to the C19=O group of the prosthetic group indicates the Z,E isomerization around the C15=C16-methine bridge of the chromophore during the formation of meta-Ra. In the difference spectra of the parent states only small differences are observed in this region suggesting that the frequency of the carbonyl group is similar in Pr and Pfr. Since the FT-IR difference spectra between lumi-F and Pfr show great similarities to the spectra of the parent states, it is assumed that during the formation of lumi-F the chromophore largely returns into the primary Pr conformation. The FT-IR spectra recorded in a medium of 2H2O generally show a downshift of the significant bands due to the isotope effect. The appearance of a characteristic band around 935 cm-1 in all 2H2O spectra suggests an assignment to an N-2H bending vibration of the chromophore.     

Cloning and expression of thermostable beta-glycosidase gene from Thermus nonproteolyticus HG102 and characterization of recombinant enzyme

The gene coding for beta-glycosidase (EC 3.2.1.21) from Thermus nonproteolyticus HG102 was cloned and expressed in Escherichia coli. The gene open reading frame was 1311 bp, and it codes for 437 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of the glycosyl hydrolase family I. The enzyme had high content of Arg and Pro. The recombinant enzyme was purified to homogeneity with heat precipitation, ammonium sulfate precipitation, DEAE-cellulose (DE52) chromatography, and prepared slab polyacrylamide gel electrophoresis. The enzyme was monomeric and had a molecular mass of 48,900 Daltons and a pI of 5.2. The enzyme showed optimum activity at pH 5.6 and 90 degrees C. It catalyzed the hydrolysis of beta-D-glucoside, beta-D-galactoside, beta-D-fucoside, and beta-D-mannoside. Lineweaver-Burk plots showed that the kcat/Km ratio for beta-D-glucoside and beta-D-fucoside was higher than for beta-D-mannoside and beta-D-galactoside. The enzyme was extremely thermostable, with a half-life of 2.5 h at 90 degrees C, and was stable over a wide range of pH. It also had transglycosidic activity at high temperature.     

The photoreactions of recombinant phytochrome CphA from the cyanobacterium Calothrix PCC7601: a low-temperature UV-Vis and FTIR study

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV–Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV–Vis and FTIR experiments. Three intermediates have been trapped at −25, −45 and −120°C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at −70 and −140°C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore–protein interactions.     

De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae

In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos‐aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N‐terminal sulfonation of tryptic peptides by 4‐sulfophenyl isothiocyanate (SPITC) and MALDI‐TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC‐derivatized peptides underwent facile fragmentation, predominantly resulting in y‐series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both α‐ and β‐phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI‐ and ESI‐MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species. Copyright © 2008 John Wiley & Sons, Ltd.     

Construction, expression, and characterization of recombinant hirudin in Escherichia coli

The mutant gene of HV2-K47 was obtained by polymerase chain reaction-directed mutagenesis and expressed in Escherichia coli. Many elements that could affect its expression level were compared. The product was purified to homogeneity via three chromatographic steps—ion exchange, gel filtration, and reverse phase chromatography—on the AKTA Explorer System. The antithrombin activity of HV2-K47 is much higher than that of recombinant HV2. Some properties and expression conditions were investigated systematically, which would be useful for further studies of hirudin and other small proteins.     

Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase

A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.     

Induction of the synthesis of an UV-absorbing substance in the green alga Prasiola stipitata

The chlorophyte Prasiola stipitata produces a UV-absorbing substance with an absorption maximum at 324 nm. The wavelength-dependent induction of the synthesis of this substance was investigated using simulated solar radiation in combination with 15 cut-off and one broad-band filter. The algae were exposed from three different distances (89, 100 and 119 cm) to the solar simulator producing a maximum of 203.58, 1.24 and 46.86 W/m(2) and a minimum of 107.94, 0.64 and 24.44 W/m(2) irradiances for PAR, UV-B and UV-A, respectively. A polychromatic action spectrum was calculated from the pooled results showing a clear maximum at 300 nm in the long-wavelength UV-B range, but there is still some induction caused by UV-A and PAR. The ratio of the effectiveness from PAR to UV-A to UV-B amounts to 1:2:22.     

Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease

We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.     

Purification and characterization of recombinant tropomyosins

The cloning of a cDNA coding for the skeletal human beta-tropomyosin in the bacterial expression vector pKK233-2 is reported. Deletion mutants were also constructed. pCF-T1088 was obtained by elimination of exon 9 and pCF-T1089 was built by deleting 2/3 of the first exon. The recombinant tropomyosins were synthesized in E. coli after induction by IPTG. The mutant proteins were characterized by western blot using antibodies raised against native tropomyosin. The amount of the human protein synthesized in E. coli varies with each mutant, suggesting the involvement of the structure of the protein or of the mRNA on the synthesis or the stability of the recombinant protein. After precipitation of most of the bacterial proteins at 100 degrees C, purification was achieved by high-performance liquid chromatography (HPLC) using TSK-DEAE, hydroxyapatite and reversed-phase columns. The chromatographic behaviour of the mutants were compared. Characterization of the mutated tropomyosins was achieved by tryptic digestion and analysis of the peptide composition by reversed-phase HPLC. A computer program for predicting the retention times of the peptides generated was written. It is shown that it is possible to identify the mutations solely by comparing the chromatogram of the tryptic digest with the profile obtained by computer simulation.     

Scalarin,a new pentacyclic C-25 terpenoid from the sponge Cacospongia scalaris

On the basis of chemical and physico-chemical evidence, structure I has been assigned to scalarin, a new C-25 terpenoid isolated from the marine sponge Cacospongia scalaris.     

Cloning and characterization of an active fragment of luciferase from a luminescent marine alga, Pyrocystis lunula

Two marine dinoflagellates, Lingulodinium polyedrum and Pyrocystis lunula, emit light in a reaction involving the enzymatic oxidation of its tetrapyrrole luciferin by molecular oxygen. The characteristic properties of P. lunula luciferase have not been clarified, whereas L. polyedrum luciferase, which has three active domains, has been characterized. A cloned partial cDNA of the P. lunula luciferase encodes an active fragment corresponding to part of domain 2 and all of domain 3 of L. polyedrum luciferase. The homology of the amino acid sequence between the two luciferases in domain 3 is about 84.3%. A recombinant His-tagged luciferase fragment containing domain 3 (Mr = 46 kDa) catalyzed the light-emitting oxidation of luciferin (lambdamax = 474 nm). This protein was purified by a single affinity-chromatography procedure. The pH-activity profile and the bioluminescence spectrum of the recombinant enzyme having a third domain are almost identical to those of an extract from P. lunula cultured in vitro. The recombinant enzyme is active at pH 8.0, although the recombinant enzyme derived from the second domain of L. polyedrum luciferase is inactive at pH 8.0. Substitution of Glu-201 by histidine in the third domain of P. lunula luciferase showed a decrease of activity above pH 7.0, suggesting that histidine residues could be responsible for pH-sensitivity in dinoflagellate luciferase.     

Heterologous expression, biosynthesis, and mutagenesis of type II lantibiotics from Bacillus licheniformis in Escherichia coli

Lichenicidin is a class II two-component lantibiotic produced by Bacillus licheniformis. It is composed of the two peptides Bliα and Bliβ, which act synergistically against various Gram-positive bacteria. The lichenicidin gene cluster was successfully expressed in Escherichia coli, thus constituting the first report to our knowledge of a full reconstitution of a lantibiotic biosynthetic pathway in?vivo by a Gram-negative host. This system was further exploited to characterize and assign the function of proteins encoded in the biosynthetic gene cluster in the maturation of lichenicidin peptides. Moreover, a trans complementation system was developed for expression of Bliα and Bliβ variants in?vivo. This contribution will spur future studies in the heterologous expression and engineering of lantibiotics.     

Intracellular release of recombinant green fluorescent protein (gfp uv ) from Escherichia coli

The recombinant green fluorescent protein (gfp _uv ) was expressed by Escherichia coli DH5-α cells transformed with the plasmid pGFPuv. The gfp _uv was selectively permeabilized from the cells in buffer solution (25 mM Tris-HCl, pH 8.0), after freezing (−70°C for 15 h), by four freeze (−20°C)/thaw cycles interlaid by sonication. The average content of released gfp _uv (experiment 2) was 7.76, 34.58, 39.38, 12.90, and 5.38%, for the initial freezing (−70°C) and the first, second, third and fourth freeze/thaw cycles, respectively. Superfusion on freezing was observed between −11°C and −14°C, after which it reached −20°C at 0.83°C/min.     

Proficiency tests using four batches of green alga with controlled levels of cadmium

A set of test materials of the green alga Chlorella vulgaris with different levels of naturally bound Cd and about the same levels of other 13 essential or trace elements (BIOMA 1–4) was produced for proficiency testing of laboratories involved in elemental analysis of food. Criteria of the laboratories’ performance, such as the z-score and combination scores RSZ and SSZ were evaluated and discussed in terms of their suitability for proficiency testing using a set of test materials with the same matrix. The use of the test materials as a set of internal reference materials has also been suggested. Received: 23 May 1997 / Revised: 1 August 1997 / Accepted: 11 August 1997