Paragonimus westermani: molecular cloning,expression, and characterization of a recombinant yolk ferritin

Ferritin is an intracellular protein involved in iron metabolism. A cDNA PwYF-1 cloned from the adult Paragonimus westermani cDNA library encoded a putative polypeptide of 216 amino acids homologous with ferritins of vertebrates and invertebrates. Febinding motifs identified in PwYF-1 polypeptide were conserved and predicted to form a ferroxidase center. PwYF-1 polypeptide contained an extended peptide of 45 amino acids at its C-terminus. Recombinant PwYF-1 protein, expressed and purified from Escherichia coli, showed iron-uptake ability and ferroxidase activity. Ferroxidase activity of recombinant PwYF-1 protein was reactivated by secondary addition of apotransferrin to assay mixture. Mouse immune serum raised against the recombinant PwYF-1 protein recognized specifically 24 kDa protein from adult P. westermani lysate. PwYF-1 protein was localized to vitelline follicles and the eggs of P. westermani. Collectively, PwYF-1 protein was identified as a P. westermani yolk ferritin.     

Constituents from the stems of Actinodaphne lancifolia

Two C(16)-lactonic compounds, actinolides A-B (1-2), were isolated from the stems of Actinodaphne lancifolia, together with five known lactones (3-7) and three known lignans (8-10). Their structures were determined spectroscopically, which included 2D NMR spectroscopic analysis.     

Diversity and varietal classification of Hibiscus syriacus L. with the heterogeneity within retrotransposon-like elements

Retrotransposons are present in multi-copy numbers that are integrated into plant genomes with considerable heterogeneous sequences within a single plant and between plant species, which allows the use of retrotransposons as additional sources of DNA polymorphism. A primer design for the sequence-tagged specific site and cleaved amplified polymorphic sequences (STS-CAPs) that are derived from retrotransposon-like sequences was developed for the molecular marker analysis in Hibiscus syriacus. This method was applied for the detection of sequence variations of intact retrotransposons that exist in plant genomes, which resulted in higher polymorphisms than in the amplified fragment length polymorphism (AFLP). Through STS-CAPs, specific fingerprinting data among H. syriacus varieties can be easily distinguished and generated with reproducible results. It could also be adapted to any species that possess multi-copy retrotransposons for varietal identification as well as the assessment of genetic relationships.     

Activities of antioxidant and redox enzymes in human normal hepatic and hepatoma cell lines

The cellular defense system (including glutathione, glutathione-related enzymes, antioxidant and redox enzymes) plays a crucial role in cell survival and growth in aerobic organisms. To understand its physiological role in tumor cells, the glutathione content and related enzyme activities in the human normal hepatic cell line, Chang and human hepatoma cell line, HepG2, were systematically measured and compared. Superoxide dismutase, catalase, and glutathione peroxidase activities are 2.8-, 4.3-, and 2.9-fold higher in HepG2 cells than in Chang cells. Total glutathione content is also about 1.4-fold higher in HepG2, which is supported by significant increases in gamma-glutamylcysteine synthetase and glutathione synthetase activities. Two other glutathione-related enzymes, glutathione reductase and gamma-glutamyltranspeptidase, are upregulated in HepG2 cells. However, thioredoxin reductase and glutathione S-transferase activities are significantly lower in HepG2 cells. These results propose that defense-related enzymes are largely modulated in tumor cells, which might be linked to their growth and maintenance.     

A direct repeat of N-type Ca2+ channel alpha1B gene functions as a negative regulatory element in HeLa cells

The expression of the N-type voltage-gated calcium channel alpha1B gene is restricted to neurons by a 5'-upstream region (-3992 to -1788) that contains negative regulatory element(s) that are active in non-neuronal cells. A 39 bp DNA element, which is repeated nine times in a head-to-tail fashion, was found within the same region. To examine whether this direct repeat (DR) may function as a negatively acting cis-regulatory element, several fusion plasmids, DR-110alpha1BLUC (1X), DR-SV40LUC (IX, 2X), in which one or two copies of the DR fragment were subcloned upstream of the homologous and heterologous promoters, were transiently transfected into HeLa and NS20Y cells. The promoter activity of DR-110alpha1BLUC (1X) decreased to approximately 17% of the 110alph(a1B)LUC construct in HeLa cells. The expression of the DR-SV40LUC (1X) and DR-SV40LUC (2X) plasmids was also reduced to 50 to 23% of the levels that were observed in the pGL2-Promoter in the same cells. However, no repression of the DR constructs was observed in NS20Y cells. An electrophoretic mobility shift assay showed that two DR-specific complexes were detected in HeLa cells, but not in NS20Y cells. In addition, Southwestern blotting revealed the presence of approximately 33 and 43 kDa proteins in HeLa cells. Overall, these results suggest that a 39 bp DNA element might act as repressor in non-neuron cells through the specific interactions of the DNA-proteins.     

Spectroscopic studies of peroxyacetic acid reaction intermediates of cytochrome P450cam and chloroperoxidase

It is generally assumed that the putative compound I (cpd I) in cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I of peroxidases and catalases and many synthetic cpd I analogues. In these systems one oxidation equivalent resides on the Fe(IV)=O unit (d(4), S=1) and one is located on the porphyrin (S'=1/2), constituting a magnetically coupled ferryl iron-oxo porphyrin pi-cation radical system. However, this laboratory has recently reported detection of a ferryl iron (S=1) and a tyrosyl radical (S'=1/2), via M?ssbauer and EPR studies of 8 ms-reaction intermediates of substrate-free P450cam from Pseudomonas putida, prepared by a freeze-quench method using peroxyacetic acid as the oxidizing agent [Schünemann et al., FEBS Lett. 479 (2000) 149]. In the present study we show that under the same reaction conditions, but in the presence of the substrate camphor, only trace amounts of the tyrosine radical are formed and no Fe(IV) is detectable. We conclude that camphor restricts the access of the heme pocket by peroxyacetic acid. This conclusion is supported by the additional finding that binding of camphor and metyrapone inhibit heme bleaching at room temperature and longer reaction times, forming only trace amounts of 5-hydroxy-camphor, the hydroxylation product of camphor, during peroxyacetic acid oxidation. As a control we performed freeze-quench experiments with chloroperoxidase from Caldariomyces fumago using peroxyacetic acid under the identical conditions used for the substrate-free P450cam oxidations. We were able to confirm earlier findings [Rutter et al., Biochemistry 23 (1984) 6809], that an antiferromagnetically coupled Fe(IV)=O porphyrin pi-cation radical system is formed. We conclude that CPO and P450 behave differently when reacting with peracids during an 8-ms reaction time. In P450cam the formation of Fe(IV) is accompanied by the formation of a tyrosine radical, whereas in CPO Fe(IV) formation is accompanied by the formation of a porphyrin radical.     

A chimeric Anabaena/ Escherichia coli KdpD protein (Anacoli KdpD) functionally interacts with E. coli KdpE and activates kdp expression in E. coli

The kdpFABC operon, coding for a high-affinity K(+)-translocating P-type ATPase, is expressed in Escherichia coli as a backup system during K(+) starvation or an increase in medium osmolality. Expression of the operon is regulated by the membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE. From a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31, a kdpDgene was cloned (GenBank accession no. AF213466) which codes for a KdpD protein (365 amino acids) that lacks both the transmembrane segments and C-terminal transmitter domain and thus is shorter than E. coli KdpD. A chimeric kdpD gene was constructed and expressed in E. coli coding for a protein (Anacoli KdpD), in which the first 365 amino acids of E. coli KdpD were replaced by those from Anabaena KdpD. In everted membrane vesicles, this chimeric Anacoli KdpD protein exhibited activities, such as autophosphorylation, transphosphorylation and ATP-dependent dephosphorylation of E. coli KdpE, which closely resemble those of the E. coli wild-type KdpD. Cells of E. coli synthesizing Anacoli KdpD expressed kdpFABC in response to K(+) limitation and osmotic upshock. The data demonstrate that Anabaena KdpD can interact with the E. coliKdpD C-terminal domain resulting in a protein that is functional in vitro as well as in vivo.     

Amino acid replacements in transmembrane domain 1 influence osmosensing but not K<Superscript>+</Superscript> sensing by the sensor kinase KdpD of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Expression of the kdpFABC operon coding for the high affinity K+ -translocating KdpFABC complex of Escherichia coli is induced by K+ limitation or high osmolality. This process is controlled by the sensor kinase/response regulator system KdpD/KdpE. To study the importance of the transmembrane domains of KdpD for stimulus perception, each amino acid residue of the transmembrane domain 1 and Asp-424 of the adjacent periplasmic loop were replaced with Cys in a KdpD derivative devoid of native Cys residues. In vivo analysis of KdpD proteins with a single Cys residue revealed that 14 out of 18 amino acid replacements caused an altered response towards an osmotic upshift imposed by NaCl, whereby only four replacements also altered the response towards changes in the K+ concentration. The in vitro activities of most of the KdpD derivatives were in the range of KdpD devoid of native Cys residues. The results reveal that the osmosensing and K+ -sensing properties of KdpD can be dissected. Furthermore, the data support the hypothesis that osmosensing involves amino acid residues of the transmembrane domains.