Influencing factors on the safety and effectiveness of venom immunotherapy.

BACKGROUND AND OBJECTIVE: The safety profile of venom immunotherapy (VIT) is a relevant issue and considerable differences in safety and efficacy of VIT have been reported. The primary aim of this study was to evaluate the safety of ACE inhibitors and beta-blockers during VIT, which has already been published. For a second analysis, data concerning premedication and venom preparations in relation to systemic adverse events (AE) during the up-dosing phase and the first year of the maintenance phase were evaluated as well as the outcome of field stings and sting challenges. METHODS: The study was conducted as an open, prospective, observational, multicenter study. In total, 1,425 patients were enrolled and VIT was performed in 1,342 patients. RESULTS: Premedication with oral antihistamines was taken by 52.1% of patients during the up-dosing and 19.7% of patients during the maintenance phase. Taking antihistamines had no effect on the frequency of systemic AE (p=0.11) but large local reactions (LLR) were less frequently seen (OR: 0.74; 95% CI: 0.58-0.96; p=0.02). Aqueous preparations were preferentially used for up-dosing (73.0%) and depot preparations for the maintenance phase (64.5%). The type of venom preparation neither had an influence on the frequency of systemic AE nor on the effectiveness of VIT (p=0.26 and p=0.80, respectively), while LLR were less frequently seen when depot preparations were used (p<0.001). CONCLUSION: Pretreatment with oral antihistamines during VIT significantly reduces the frequency of LLR but not systemic AE. All venom preparations used were equally effective and did not differ in the frequency of systemic AE.

The spleen accumulates advanced glycation end products in the chicken: tissue comparison made with rat.

Glycation starts from nonenzymatic amino-carbonyl reaction that binds carbonyl group of reducing sugars to the amino group of amino acids. Glycation leads to further complex reactions to form advanced glycation end products (AGE). Because AGE are implicated in the gradual development of diabetic complications, tissue accumulation of AGE has been widely examined in various tissues of rats. Avian species are known to have high body temperature and blood glucose concentration compared with mammals. Although these characteristics enabled chickens to be used as experimental models for diabetes mellitus, the information of AGE accumulation in various tissues of chickens has not been limited so far. In the present study, therefore, the radioactive AGE prepared by reacting (14)C-glucose and amino acids were intravenously administrated, and comparison of tissue accumulation of (14)C-labeled AGE was made between chickens and rats. At 30 min after administration, tissues (18-20) were collected, and the radioactivity incorporated into tissues was determined. High levels of radioactivity per gram of tissue in the liver and kidney were observed in both rats and chickens. In chickens but not rats, a large amount of (14)C-labeled AGE incorporated into 1 g of spleen was observed, and the specific accumulation of AGE in the avian spleen might have a particular role in immune response in avian species.

Cryopreservation of nematode Caenorhabditis elegans in the adult stage.

Cryopreservation of nematode Caenorhabditis elegans in the adult stage is of importance as the nematode is a powerful research model organism. In this study, we applied the protocol previously established for cryopreservation of the L4 nematode to the adult one, and achieved a survival rate of 84%. When ice seeding was induced with bacteria P. syringae directly added to the nematode suspension instead of using a pre-cooled steel sticking needle, comparable survival rate was obtained after thawing. Moreover, a simple freezing device composed of a polystyrene foam box surrounded by a Dewar vessel put in a deep freezer was developed for a practical use. This simple method obtained a survival rate of 69 ± 4% for the adult nematode after thawing.

Dietary carbohydrate influences the colocalization pattern of Glucagon-like Peptide-1 with neurotensin in the chicken ileum.

Glucagon-like peptide (GLP)-1 colocalizes with neurotensin (NT) in the same enteroendocrine cells (EECs) of the chicken ileum. The present study was designed to clarify the influence of dietary carbohydrate (CHO) on the colocalization pattern of GLP-1 with NT in the chicken distal ileum. Male White Leghorn chickens at 6 weeks of age (n = 15) were divided into three groups, a control and two experimental (low-CHO and CHO-free), with five chickens in each, and fed control or experimental diets for 7 d. Distal ileum was collected from each bird as a tissue sample and subjected to double immunofluorescence staining to detect GLP-1 and NT. Three types of EEC, GLP-1+/NT+, GLP-1+/NT- and GLP-1-/NT+, were demonstrated in the chicken ileum. GLP-1+/NT+ cells in the control group had a spindle-like shape with a long cytoplasmic process, but those in the experimental groups were round and lacked a cytoplasmic process. The ratio of GLP-1+/NT+ cells was significantly decreased in the two experimental groups compared with that in the control group. The ratio of GLP-1+/NT+ cells was significantly lower than those of GLP-1+/NT- and GLP-1-/NT+ cells in the two experimental groups. Most cells that were immunoreactive for GLP-1 and NT antisera lacked signals of proglucagon (PG) and NT precursor (NTP) mRNA in the experimental groups. The number of EECs expressing PG and NTP mRNA signals showed tendencies for decreases with a reduction of dietary CHO level. These findings suggest that dietary CHO could be a significant regulator of the pattern of colocalization pattern of GLP-1 with NT in the chicken ileum.

Pre-clinical evaluation of liposomal gene transfer to improve dermal and epidermal regeneration.

Liposomal gene transfer effectively enhances dermal and epidermal regeneration in burned rodents. To advance this treatment to clinical studies, we investigated the efficacy of liposomal gene transfer in a clinically relevant porcine wound model. Mimicking the clinical scenario, six female Yorkshire pigs (40-50 kg) received up to 12 burns of 50 cm(2) area that were fully excised and covered with skin autograft meshed at 4:1 ratio 24 h post-burn. Animals received control injections (empty liposomes), liposomes (DMRIE-C) containing 1 mg LacZ-cDNA, or liposomes (DMRIE-C) with 1 mg of platelet-derived growth factor (PDGF)-cDNA, or the naked PDGF gene. Serial biopsies were taken from different wound sites at multiple time points up to 12 days post-wounding. Transfection efficacy and transfection rate of LacZ and localization of beta-gal were determined by immunohistochemical and immunofluorescent techniques. RT-PCR and multiplex protein analysis (ELISA) were used to measure levels of growth factor mRNA transcribed and growth factor protein translated. Wound re-epithelialization and graft adhesion was evaluated using planimetric analysis and clinical scores. We found that peak transfection of liposomal beta-galactosidase occurred on day 2, with a fluorescence increase of 154% to baseline (P<0.001). Transfection intensity dropped to 115% above baseline on day 4 (P<0.001) and 109% on day 7. Immunohistochemistry showed a maximum transfection rate of 34% of cells in wound tissue. Gene transfer of liposomal PDGF-cDNA resulted in increased PDGF-mRNA and protein expression on days 2 and 4, and accelerated wound re-epithlialization as well as graft adhesion on day 9 (P<0.05). In this study, we showed that liposomal cDNA gene transfer is possible in a porcine wound model, and by using PDGF-cDNA we further showed that dermal and epidermal regeneration can be improved. These data indicate that liposomal gene transfer can be a new therapeutic approach to improve wound healing in humans.

Impact of training of mothers, drug shop attendants and voluntary health workers on effective diagnosis and treatment of malaria in Lagos, Nigeria.

BACKGROUND: The National Malaria Eradication Program and international agencies are keen on scaling up the use of malaria rapid diagnostic tests (mRDTs) and artemisinin-based combination therapies (ACTs) for effective diagnosis and treatment of the disease. However, poor diagnostic skills and inappropriate treatment are limiting the efforts. In Nigeria, a large proportion of infected patients self-diagnose and treat while many others seek care from informal drug attendants and voluntary health workers. AIMS: This study describes the impact of training voluntary health workers, drug shop attendants, and mothers on effective case detection and treatment of malaria in Lagos, Nigeria. METHODS: We trained mothers accessing antenatal care, drug shop attendants, and voluntary health workers selected from the three districts of Lagos, on the use of histidine-rich protein-2-based mRDTs and ACTs. Pre- and post-training assessments, focus group discussions (FGDs), and in-depth interviews (IDIs) were carried out. RESULTS: The knowledge, attitude, and skill of the participants to achieve the goal of "test, treat, and track" using mRDT and ACTs were low (11%-55%). There was a low awareness of other non-malaria fevers among mothers. Self-medication was widely practiced (31.3%). FGDs and IDIs revealed that health-care providers administered antimalarials without diagnosis. Training significantly improved participants' knowledge and expertise on the use of mRDTs and ACTs (P = 0.02). The participants' field performance on mRDT use was significantly correlated with their category (bivariate r = 0.51, P = 0.001). There was no statistically significant association between the participants' level of education or previous field experience and their field performance on mRDT (r = 0.12, P = 0.9; χ 2= 38, df = 2 and P = 0.49). CONCLUSION: These findings suggest that training of stakeholders in malaria control improves diagnosis and treatment of malaria. However, a broader scope of training in other settings may be required for an effective malaria control in Nigeria.

Clinical and prognostic significance of cytokine receptor expression in adult acute lymphoblastic leukemia: interleukin-2 receptor alpha-chain predicts a poor prognosis.

We quantitatively assessed the expression of cytokine receptors (interleukin-2 receptor (IL-2R), IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, granulocyte-macrophage colony-stimulating factor R (GM-CSFR), G-CSFR, c-fms, c-mpl, c-kit and FLT3) in cells from 211 adults with acute lymphoblastic leukemia (ALL) by flow cytometry and determined their prevalence and clinical significance. Although all cytokine receptors were expressed to various degrees, the levels of IL-3R alpha-chain (IL-3Ralpha), IL-2Ralpha, IL-2Rbeta, IL-7Ralpha, common-Rgamma(gammac), c-mpl, c-kit and FLT3 exhibited a wide spectrum > or =2000 sites/cell. Among them, IL-3Ralpha, IL-2Ralpha and FLT3 were highly expressed in B-lineage ALL, whereas IL-7Ralpha, gammac and c-kit predominated in T-lineage ALL. Higher levels of IL-3Ralpha, IL-2Ralpha, c-kit and FLT3 correlated with the expression of CD13/33. Increased IL-2Ralpha levels related to the presence of Philadelphia chromosome (Ph), leukocytosis and shorter event-free survival (EFS). C-kit preferred in male. Elevated FLT3 levels correlated with age > or =60 years. Multivariate analysis in B-lineage ALL revealed only IL-2Ralpha (P=0.028) and Ph (P=0.020) as independent factors for EFS. These findings suggest that several cytokine receptors associated with certain cellular and clinical features, but IL-2Ralpha solely had a prognostic value and should be considered as a major prognostic factor for adult ALL that is comparable with Ph.

Phenotypic conversion of T lymphoblastic lymphoma to acute biphenotypic leukemia composed of lymphoblasts and myeloblasts. Molecular genetic evidence of the same clonal origin.

Acute biphenotypic leukemia composed of lymphoblasts and myeloblasts developed in a patient with T lymphoblastic lymphoma (T-LBL) who had an anterior mediastinal mass. A novel myeloid cell line, termed TK-1, has been established from his peripheral blood after the leukemic conversion. The identical rearranged pattern of T cell receptor gamma-chain gene was observed among the DNAs derived from lymph node cells in the lymphoma phase, the myeloid cell line TK-1, and the subclones with different karyotypes (TK-1B and TK-1D), which showed that myeloid cells had been derived from the T-LBL of the same patient. This finding demonstrates that phenotypic conversion occurs in the clonally propagating tumor cells and suggests that some hematopoietic cells retain the capacity to adopt either lineage.

Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR inhibitor. These data reveal that biomarker-directed trials for FGFR1-amplified SCC require assessment of FGFR1 protein expression and uncover novel therapeutic strategies for FGFR1-amplified SCC with low FGFR1 protein expression.

Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa.

Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.

Identification of amino-acid residues linked to different properties of phosphoribosylpyrophosphate synthetase isoforms I and II.

The catalytic subunit of rat liver phosphoribosylpyrophosphate synthetase is composed of two isoforms, PRS I and PRS II. The amino-acid sequences differ only by 13 residues, out of which two Lys residues of PRS I at positions 4 and 152 give net additional positive charges to PRS I. Previous work has shown that PRS I is more sensitive to inhibition by ADP and GDP and more stable to heat treatment than is PRS II. To identify amino-acid residues responsible for the different properties, five chimeric enzymes between rat PRS I and PRS II and two mutated enzymes with a single point mutation at position 152 were constructed; these enzymes were produced in Escherichia coli. Changing Lys-4 of PRS I to Val, together with Ile-5 to Leu, completely abolished sensitivity to GDP inhibition of PRS I, indicating that Lys-4 in PRS I is critical for GDP inhibition. The substitutions at position 152 had little effect on GDP inhibition. Characterization of the chimeric enzymes revealed that residues between residues 54-110 and 229-317, namely, Val-55 and/or Ala-81, and Arg-242 and/or Cys-264 of PRS I also contribute to the strong GDP inhibition. Lys-4 was also important for the strong ADP inhibition of PRS I. Regarding the physical properties, chimeric enzymes bearing residues 12-53 of PRS I were stable at 49 degrees C and with digestion with papain and proteinase K. Our observations suggest that Lys-17, Ile-18, and/or Cys-40 of PRS I contribute to stability of the enzyme.

Variations in the C-terminal repeats of the knob-associated histidine-rich protein of Plasmodium falciparum.

The knob-associated histidine rich protein (KAHRP) of Plasmodium falciparum plays an important role in the pathophysiology of cerebral malaria. In the present study, the immunogenic C-terminal repeat domain of the KAHRP gene was amplified, cloned and sequenced from the Indian (RJ181) and Honduran (HB3) isolates of P. falciparum. Based on the number and types of repeats in the domain, we report here the presence of three unique variant forms of KAHRP among these isolates. The Indian isolate (RJ181) contained four units of the decapeptide repeats whereas the Honduran isolate (HB3) contained two forms i.e. one form containing four decapeptide repeats plus a tetrapeptide subunit and the other form containing three decapeptide repeats plus a tetrapeptide subunit. Thus, all together, the number of KAHRP variants is increased to five which includes previously described two variants, each containing either 3 or 5 decapeptide repeats. This high rate of variability in the antigenic domain of the KAHRP gene via deletion or addition of whole or part of the decapeptide units could be involved in the evasion of host immune system possibly by providing the speculative complementarity to the vargene product. The results of the present study will be useful in designing the suitable molecular therapeutic reagents for cerebral malaria.

cDNA sequence of a translational elongation factor Ts homologue from Caenorhabditis elegans: mitochondrial factor-specific features found in the nematode homologue peptide.

The cDNA for a homologue of elongation factor Ts which probably functions in mitochondria has been sequenced from a nematode Caenorhabditis elegans. The deduced amino acid sequence (316 amino acids long) has a possible transit peptide sequence at the amino terminus and several common specific features for mammalian mitochondrial EF-Ts. The amino acid identities in the protein from C. elegans compared with those of bovine mitochondria and Escherichia coli are 29.5% and 24.0%, respectively. The C. elegans sequence was classified as a long EF-Ts (ca. 280 amino acids long) similar to peptides from mammalian mitochondria and eubacteria other than Thermus and cyanobacteria (except Spirulina platensis), rather than short EF-Ts (ca. 200 amino acids long) as those of Thermus, cyanobacteria and plastids.

Cloning and sequencing of rat cDNA for the 41-kDa phosphoribosylpyrophosphate synthetase-associated protein has a high homology to the catalytic subunits and the 39-kDa associated protein.

Rat liver phosphoribosylpyrophosphate synthetase is a complex aggregate of 34-kDa catalytic subunits (PRS I and II) and 39- and 41-kDa associated proteins (PAP39 and 41). When the rat cDNA encoding PAP41 was isolated, the deduced protein sequence was seen to contain 369 amino acids with a calculated molecular mass of 41130. PAP41 has a 79 and 49% identity with PAP39 and PRSs, respectively. When conservative substitutions are included, PAP41 and the three other components have a 66% homology. PAP41 shares some common features with PAP39 and the two proteins form the PAP subfamily. The mRNA of PAP41 is present in all rat tissues we examined.

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

Rat liver phosphoribosylpyrophosphate synthetase is activated by free Mg2+ in a manner that overcomes its inhibition by nucleotides.

Phosphoribosylpyrophosphate synthetase is activated by Pi and free Mg2+ as an essential activator and inhibited by nucleotides, especially ADP and GDP. The rat liver enzyme is a complex aggregate of two highly homologous catalytic subunits (PRS I and PRS II) and two associated proteins (PAP39 and PAP41). PRS I is more sensitive to inhibition by ADP and GDP than is PRS II. The native liver enzyme showed a weaker sensitivity to inhibition by nucleotides than expected from its composition. To further understand the regulation of the liver enzyme, kinetic studies of each subunit component and the liver enzyme regarding Mg2+ activation and inhibition by ADP and GDP were carried out. Assay conditions were designed to keep free Mg2+ at constant concentrations. (1) GDP, as MgGDP, did not affect the apparent Km values of PRS I for MgATP and ribose-5-phosphate but did dramatically increase the apparent Ka value for free Mg2+. (2) In contrast, ADP, as MgADP, increased the Km value for MgATP of PRS I as well as the Ka value for free Mg2+. (3) High concentrations of free Mg2+ almost completely nullified the inhibitory effect of MgGDP and partly that of MgADP on PRS I. (4) At low free Mg2+ concentrations within the physiological range, inhibition by the nucleotides is of physiological significance and conversely, variation in free Mg2+ concentrations critically affects the enzyme activity in the presence of inhibitory nucleotides. (5) The response of PRS II and the native liver enzyme is similar to that of PRS I, while the effects of MgGDP and MgADP were smaller than that on PRS I. (6) We propose that MgGDP binds to a regulatory site of PRS I and PRS II and MgADP to the substrate MgATP site and also the regulatory site. The allosteric interaction of the regulatory site and the Mg2+ binding site is also considered.

Partial reconstitution of mammalian phosphoribosylpyrophosphate synthetase in Escherichia coli cells. Coexpression of catalytic subunits with the 39-kDa associated protein leads to formation of soluble multimeric complexes of various compositions.

Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of 34-kDa catalytic subunits (PRS I and PRS II) and homologous 39- and 41-kDa proteins termed PRPP synthetase-associated proteins (PAPs). While a negative regulatory role was indicated for PAPs, the physiological function of PAPs is less well understood. We attempted to prepare recombinant 39-kDa PAP (PAP39) and to reconstitute the enzyme complex. Free PAP39 was poorly expressed in Escherichia coli, while expression of protein fused with glutathione S-transferase was successful. The purified fusion protein had no PRPP synthetase activity, and bound to dissociated PRS I and PRS II, with a similar affinity. A free form of PAP39 prepared from the fusion protein formed insoluble aggregates. The enzyme complex was then partially reconstituted in situ by coexpression of PAP39 with PRS I or PRS II in E. coli cells. This coexpression led to formation of soluble complexes of various compositions, depending on the conditions. When the relative amount of PAP39 was higher, specific catalytic activities, in terms of the amount of the catalytic subunit, were lowered. PAP39 complexed with PRS I was more readily degraded by proteolysis than seen with PRS II, in vivo and in vitro. These results provide additional, strong evidence for that PAP39 has no catalytic activity in the enzyme complex, but does exert inhibitory effects in an amount-dependent manner, and that composition of the enzyme complex varies, depending on the relative abundance of components present at the site of aggregate formation.

Electron-transfer complexes of Ascaris suum muscle mitochondria. III. Composition and fumarate reductase activity of complex II.

Complex II of the anaerobic respiratory chain in Ascaris muscle mitochondria showed a high fumarate reductase activity when reduced methyl viologen was used as the electron donor. The maximum activity was 49 mumol/min per mg protein, which is much higher than that of the mammalian counterpart. The mitochondria of Ascaris-fertilized eggs, which require oxygen for its development, also showed fumarate reductase activity with a specific activity intermediate between those of adult Ascaris and mammals. Antibody against the Ascaris flavoprotein subunit reacted with the mammalian counterparts, whereas those against the Ascaris iron-sulfur protein subunit did not crossreact, although the amino acid compositions of the subunits in Ascaris and bovine heart were quite similar. Cytochrome b-558 of Ascaris complex II was separated from flavoprotein and iron-sulphur protein subunits by high performance liquid chromatography with a gel permeation system in the presence of Sarkosyl. Isolated cytochrome b-558 is composed of two hydrophobic polypeptides with molecular masses of 17.2 and 12.5 kDa determined by gradient gel, which correspond to the two small subunits of complex II. Amino acid compositions of these small subunits showed little similarity with those of cytochrome b-560 of bovine heart complex II. NADH-fumarate reductase, which is the final enzyme complex in the anaerobic respiratory chain in Ascaris, was reconstituted with bovine heart complex I, Ascaris complex II and phospholipids. The maximum activity was 430 nmol/min per mg protein of complex II. Rhodoquinone was essential for this reconstitution, whereas ubiquinone showed no effect. The results clearly indicate the unique role of Ascaris complex II as fumarate reductase and the indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase system.

Developmental changes in the respiratory chain of Ascaris mitochondria.

The Ascaris larval respiratory chain, particularly complex II (succinate-ubiquinone oxidoreductase), was characterized in isolated mitochondria. Low-temperature difference spectra showed the presence of substrate-reducible cytochromes aa3 of complex IV, c+c1 and b of complex III (ubiquinol-cytochrome c oxidoreductase) in mitochondria from second-stage larvae (L2 mitochondria). Quinone analysis by high-performance liquid chromatography showed that, unlike adult mitochondria, which contain only rhodoquinone-9, L2 mitochondria contain ubiquinone-9 as a major component. Complex II in L2 mitochondria was kinetically different from that in adult mitochondria. The individual oxidoreductase activities comprising succinate oxidase, and fumarate reductase were determined in mitochondria from L2 larvae, from larvae cultured to later stages, and from adult nematodes. The L2 mitochondria exhibited the highest specific activity of cytochrome c oxidase, indicating that L2 larvae have the most aerobic respiratory chain among the stages studied. The Cybs subunit of complex II in L2 and cultured-larvae mitochondria exhibited different reactivities against anti-adult Cybs antibodies. Taken together, these results indicate that the complex II of larvae is different from its adult counterpart. In parallel with this change in mitochondrial biogenesis, biosynthetic conversion of quinones occurs during development in Ascaris nematodes.

Cloning of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122, and characterization of its gene product.

A DNA fragment that carries the gene coding for poly(3-hydroxybutyrate) (PHB) depolymerase was cloned from the chromosomal DNA of Alcaligenes faecalis AE122 isolated from seawater. The open reading frame encoding the precursor of the PHB depolymerase was 1905 base pairs (bp) long, corresponding to a protein of 635 amino acid residues (M(r) = 65,208). The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the gene, and the sequence adhering to the ribosome-binding sequence was found in front of the gene. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 28 onwards. Analysis of the deduced amino acid sequence revealed the domain structure of the protein; a signal peptide of 27 amino acids long was followed by a catalytic domain of about 400 amino acids, a fibronectin type III module sequence, and a putative substrate binding domain. The molecular mass (62,526) of the mature protein deduced from the nucleotide sequence was significantly lower than the value (95 kDa) estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but coincided well with the value (62,426) estimated from matrix-assisted laser desorption ionization mass spectra. By comparison of the primary structure with those of other PHB depolymerases, the substrate binding domain was found to consist of two domains, PHB-specific and poly(3-hydroxyvalerate)-specific ones, connected by a linker region. The PHB depolymerase gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified from culture broth and showed the same catalytic properties as the enzyme from A. faecalis.