Vascular endothelial growth factor as a predictive marker for POEMS syndrome treatment response: retrospective cohort study.

OBJECTIVE: POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes) syndrome is a rare multisystem disease characterised by plasma cell dyscrasia and overproduction of vascular endothelial growth factor (VEGF). VEGF is assumed to be useful in monitoring disease activity, because VEGF levels usually decrease after treatment. However, there is no study to investigate whether the extent of decrease in VEGF correlates with clinical outcome. We tested the predictive efficacy of serum VEGF levels in POEMS syndrome. METHOD: This was an institutional review board approved retrospective observational cohort study of 20 patients with POEMS monitored regularly for more than 12 months (median follow-up, 87 months) after treatment onset using our prospectively accumulated database of POEMS from 1999 to 2015. Patients were treated by autologous peripheral blood stem cell transplantation or thalidomide administration. Serum VEGF was measured by ELISA. Outcome measures included clinical and laboratory findings and relapse-free survival. RESULTS: Serum VEGF levels decreased rapidly after treatment, and stabilised by 6 months post treatment. Patients with normalised serum VEGF levels (<1040 pg/mL) at 6 months showed prolonged relapse-free survival (HR=12.81, 95% CI 2.691 to 90.96; p=0.0001) and greater later clinical improvement. The rate of serum VEGF reduction over the first 6 months post treatment correlated with increased grip strength, serum albumin levels, and compound muscle action potential amplitudes at 12 months. CONCLUSIONS: Serum VEGF level at 6 months post treatment is a predicative biomarker for disease activity and prognosis in POEMS syndrome. Serum VEGF could be used as a surrogate endpoint for relapse-free survival or clinical or laboratory improvement of POEMS syndrome for clinical trials.

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.

Use of the exogenous Drosophila octopamine receptor gene to study Gq-coupled receptor-mediated responses in mammalian neurons.

Diverse excitatory and inhibitory neuronal responses are mediated via Gq-coupled receptors, but the lack of a systematic comparison of different receptors or neurons has hindered a better understanding of these responses. Such a comparison may be provided by an exogenous receptor that is activated by compounds that have no effect on endogenous receptors. We therefore expressed an invertebrate biogenic amine receptor, the Drosophila octopamine receptor, in rat cortical neurons and compared octopamine receptor-mediated responses with those mediated by the group I metabotropic glutamate receptor, the endogenous Gq-coupled receptor in rat cortical neurons. Stimulation of either receptor did not result in a calcium response in octopamine receptor-expressing neurons, although octopamine preferentially elicited a calcium increase in octopamine receptor-expressing PC12h cells, while enhancing the neuronal depolarization-induced calcium increase and the electrical excitability. The increased excitability was caused by inward currents resulting from a reduction in the leak current, which was voltage-independent and blocked by genistein, a non-selective tyrosine kinase inhibitor. These results show that, in cortical neurons, exogenous octopamine receptor in mushroom bodies activated the same cell signaling pathway as endogenous metabotropic glutamate receptor, suggesting that the diverse neuronal responses mediated by Gq-coupled receptors are due to the properties of different neurons, rather than to the properties of the receptors.

Study of retrieved acetabular sockets made from high-dose, cross-linked polyethylene.

Although ultra-high molecular weight polyethylene (UHMWPE) has stable chemical properties, chemical degradation, such as oxidation reaction, progresses with long-term clinical use. The purpose of this study was to investigate the change in properties of polyethylene (PE) in vivo by examining retrieved UHMWPE sockets and high-dose, cross-linked PE (100 Mrad PE) sockets. Twenty retrieved sockets (including 2 100 Mrad PE sockets), which were implanted from 1970 to 1996, were used for analysis. The oxidation index of 100 Mrad PE sockets was approximately the same as that of the normal UHMWPE sockets in worn areas. These long-term clinical results indicate that 100 Mrad PE is sufficiently stable for clinical use and that free radicals would not affect progression of oxidation significantly.

Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration.

Using N2 cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol-fumarate reductase.

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans.

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.

Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit.

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

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.

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.

Evidence for involvement of Escherichia coli genes pmbA, csrA and a previously unrecognized gene tldD, in the control of DNA gyrase by letD (ccdB) of sex factor F.

The letA (ccdA) and letD (ccdB) genes of the F plasmid, located just outside the sequence essential for replication, contribute to stable maintenance of the plasmid in Escherichia coli cells. The letD gene product acts to inhibit partitioning of chromosomal DNA and cell division by inhibiting DNA gyrase activity, whereas the letA gene product acts to reverse the inhibitory activity of the letD gene product. To identify the host factor(s) involved in this process, we analyzed the mutants that escaped letD expression and their suppressor, and found that the three E. coli genes tldD, tldE and zfiA participate in the process, in addition to the groE genes we reported previously. The tldD and tldE mutations made cells tolerant for letD expression, as did groES mutations, while the mutation in the zfiA gene made tldD, tldE and groES mutants LetD sensitive. We hypothesize that these gene products are factors that modulate activity of DNA gyrase along with the letD gene product; the zfiA gene product acts to inhibit interaction between the LetD protein and the A subunit of DNA gyrase, while the tldD, tldE and groE gene products act to suppress the inhibitory activity of the zfiA gene product. The tldD, tldE, and zfiA genes are located at 70.4, 96.0 and 58.2 minutes on the E. coli chromosome, respectively, and code for proteins with relative molecular masses of 51,000, 48,000 and 6800, respectively. tldD is a novel gene, but the tldE and zfiA genes proved to be the pmbA gene (production of Microcin B17) and the csrA gene (carbon storage regulator), respectively.

Biological activities of racemomycin-B, beta-lysine rich streptothricin antibiotic, the main component of Streptomyces lavendulae OP-2.

Racemomycin-B (RM-B), the main component of Streptomyces lavendulae OP-2 which is the basis of 50% of the antibiotics produced, is a streptothricin antibiotic which contains three beta-lysine moieties in the molecule. RM-B had antimicrobial activity against plant-pathogenic microorganisms and growth-inhibitory activity against the root of Brassica rapa L. at the concentration of 50 ppm. It strongly inhibited the growth of Pseudomonas syringae pv. tabaci IFO-3508 (minimum inhibitory concentration (MIC): 0.4 microgram/ml), and also showed antifungal activity against six kinds of Fusarium oxysporum species (MIC: 0.1-2.0 micrograms/ml). The antimicrobial activity of RM-B was much stronger than those of RM-A and -C which contain, respectively, one and two beta-lysine moieties in their molecules. The above activities of RM-A, -C and -B were thus in the order of -B greater than -C greater than -A: namely, the biological activity of racemomycin compounds tended to be stronger with increase in the number of beta-lysine moieties in the molecule.

Hypotensive effects on spontaneously hypertensive rats and antifungal activity on various species of Fusarium oxysporum of diethylstilbestrol-related compounds.

The diethylstilbestrol-related compounds 3,3'-dihydroxy-alpha, beta-diethyldiphenylethane (I), diethylstilbestrol (II) and hexestrol (III) showed hypotensive effects on spontaneously hypertensive rats (SHR) and antifungal activities against all Fusarium oxysporum sp. tested. As previously reported, I had strong hypotensive action on normotensive rats at the dose of 10 mg/kg, while II and III showed weak hypotensive effects on these rats at the same dose. In this work, all three compounds also had hypotensive actions on SHR at the same dose. I showed the strongest hypotensive effect (-80.0 +/- 5.0 mmHg, 10 mg/kg, i.v.) on both SHR and normotensive rats. The three compounds also had antifungal activities against five kinds of Fusarium oxysporum sp. tested. Especially, II strongly inhibited the growth of Fusarium oxysporum f. sp. raphani IFO-9972 (minimum inhibitory concentration (MIC): 1.0 micrograms/ml).

Hemoglobin saitama or beta 117 (G19) His leads to Pro, a new variant causing hemolytic disease.

An electrophoretically silent unstable hemoglobin was found in a 23-year-old Japanese woman suffering from hemolytic anemia and jaundice. The unstable beta subunit was precipitated with p-chloromercuribenzoic acid and the globin was further purified by urea CM-52 column chromatography. Results of amino acid analysis, tryptic fingerprinting and manual Edman degradation of the abnormal beta T12B established a substitution of proline for histidine beta 117 (G19). This new abnormal hemoglobin was absent in other members of the family.