Acinetobacter quorum sensing contributes to inflammation-induced inhibition of orthopaedic implant osseointegration.

Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1ß levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1ß induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.

Oregano Oil and Its Principal Component, Carvacrol, Inhibit HIV-1 Fusion into Target Cells.

Oregano essential oil has long been known for its health-promoting benefits. Here, we report its activity against viral replication. Oregano oil was found to specifically inhibit lentiviruses, such as human and simian immunodeficiency viruses (HIV and SIV), irrespective of virus tropism, but not hepatitis C virus, adenovirus 5 (ADV5), Zika virus, and influenza (H1N1) virus. Oregano oil's most abundant components, carvacrol and its isomer, thymol, were shown to block virus-target cell fusion while not perturbing other stages of the virus life cycle. We detected changes in virus particle density, suggesting that cholesterol depletion from the HIV-1 envelope membrane reduces virus entry. Furthermore, infection was rescued by adding exogenous cholesterol. The evolution of viral resistance to carvacrol supported this mechanism of action with the identification of mutations in the viral gp41 fusion protein that counteracted cholesterol depletion. In addition, resistance to carvacrol emerged later than typically observed for other clinically used drugs, strengthening its antiviral potential. Structure-activity relationship studies revealed key motifs of carvacrol and thymol required for HIV neutralization and identified previously unknown active analogs. Carvacrol was also shown to additively cooperate with antiretroviral therapy. In sum, oregano oil and improved carvacrol and thymol analogs could be considered to supplement current HIV therapeutics.IMPORTANCE Oregano essential oil has multiple benefits in traditional medicine, cosmetics, and food industries. Carvacrol and its analog, thymol, are well-described components of oregano oil. Here, we show that these compounds inhibit HIV-target cell fusion independently of viral tropism. Our results suggest that carvacrol and thymol alter the cholesterol content of the viral membrane, blocking HIV-1 entry into the target cell. Resistance to carvacrol has selected for viruses with mutations in the viral envelope glycoprotein, gp41. This protein is known for its interaction with cholesterol present in membrane lipid rafts. Together, these results demonstrate the potential of therapies targeting the viral envelope membrane, and oregano oil is a safe supplement to antiretrovirals, potentially delaying disease progression and resistance development.

Potent suppression of HIV-1 cell attachment by Kudzu root extract.

There is a constant need to improve antiretrovirals against HIV since therapy is limited by cost, side effects and the emergence of drug resistance. Kudzu is a climbing vine from which the root extract (Pueraria lobata), rich in isoflavones and saponins, has long been used in traditional Chinese medicine for a variety of purposes, from weight loss to alcoholism prevention. Here we show that Kudzu root extract significantly inhibits HIV-1 entry into cell lines, primary human CD4+T lymphocytes and macrophages, without cell-associated toxicity. Specifically, Kudzu inhibits the initial attachment of the viral particle to the cell surface, a mechanism that depends on the envelope glycoprotein gp120 but is independent from the HIV-1 cell receptor CD4 and co-receptors CXCR4/CCR5. This activity seems selective to lentiviruses since Kudzu inhibits HIV-2 and simian immunodeficiency virus, but does not interfere with Hepatitis C, Influenza, Zika Brazil and adenovirus infection. Importantly, depending on the dose, Kudzu can act synergistically or additively with the current antiretroviral cocktails against HIV-1 and can block   viruses resistant to the fusion inhibitor Enfuvirtide. Together our results highlight Kudzu's root extract value as a supplement to current antiretroviral therapy against HIV.

Effects of once-monthly minodronate versus risedronate in osteoporosis patients with rheumatoid arthritis: a 12-month randomized head-to-head comparison.

A head-to-head comparison of once-monthly oral bisphosphonates minodronate (MIN) and risedronate (RIS) in patients with rheumatoid arthritis (RA) demonstrated that MIN has the same effect as RIS on increase in bone mineral density (BMD) and a stronger effect on inhibition of bone resorption than RIS, suggesting that MIN is a promising treatment option for osteoporosis patients with RA. INTRODUCTION: To evaluate the effect of once-monthly oral MIN in patients with RA, a prospective, randomized, open-label, head-to-head comparison with once-monthly oral RIS was conducted. METHODS: A total of 83 patients with RA were randomly assigned to either once-monthly oral MIN 50 mg (n = 42) or once-monthly oral RIS 75 mg (n = 41). Serial BMD and bone turnover markers were measured and compared between the treatment groups. RESULTS: BMD (lumbar spine, total hip, femoral neck) increased significantly after 12 months of treatment with MIN (3.8, 2.0, and 2.2%, respectively, P < 0.05) and RIS (3.6, 1.9, and 1.9%, respectively, P < 0.05). There were no significant differences between the treatment groups. Percent changes of bone turnover markers from baseline to 12 months in the MIN group were significantly greater than those in the RIS group (TRACP-5b: - 36.3 vs - 19.3%, P < 0.05; NTX: - 27.1 vs - 17.3%, P < 0.05; BAP: -30.2 vs -19.4%, P < 0.05). CONCLUSIONS: The present study of RA patients demonstrated that MIN has the same effect as RIS on increase in BMD and a stronger effect on inhibition of bone resorption than RIS. The results suggest that MIN is a promising treatment option for osteoporosis patients with RA.

Effect of kosher salt application on microbial profiles of poultry carcasses.

The effect of conventionally applied kosher salt on the microbiological profile of posteviscerated chicken carcasses obtained from a local commercial processing facility was evaluated. The broiler carcasses were divided into treatments 1 through 8. Standard sampling methods were used to evaluate Salmonella prevalence, aerobic plate counts, coliforms, generic Escherichia coli, and psychrotroph counts. Results indicate significant reductions in microbial populations in all the salted groups compared with controls. Significant reductions (1.45, 2.31, 2.81, and 1.48 log cfu/mL of rinse) were obtained for aerobic plate count (APC), coliforms, generic E. coli, and psychrotroph counts, respectively, on prechill salt-treated carcasses compared with controls. Salt-treated carcasses sampled after chilling had lower microbial populations compared with control chilled samples with significant reductions in coliforms and generic E. coli (1.25 and 1.77 log, respectively). Salt-treated samples had lower counts on APC and psychrotrophs after 10 d of refrigerated storage compared with controls. Finally, drip loss of salt-treated carcasses was lower after 24 h compared with nontreated controls. Based on the results, it can be concluded that salting process is an effective contributor to microbial reductions during processing that needs further investigation as a possible intervention in commercial poultry processing settings.

Skeletal muscle-secreted myokine interleukin-6 induces white adipose tissue conversion into beige adipose tissue in myostatin gene knockout pigs.

Myostatin (MSTN) is primarily expressed in skeletal muscle and plays an important role in the regulation of muscle growth and development as well as fat deposition; however, little is known about the molecular mechanism through which MSTN regulates body fat deposition. Therefore, in this study, we sought to identify the signaling pathways through which MSTN regulates fat accumulation in pigs. MSTN knockout (MSTN-/-) pigs showed increased muscle mass, decreased fat mass, and a leaner body composition. In this study, we found that the adipose tissue of MSTN-/- pigs exhibits the characteristics of beige adipose tissue, and the mRNA expression levels of beige adipose marker genes, including UCP3, Cidea, and CD137, were significantly increased. Remarkably, the observed beige phenotype was not adipocyte autonomous but rather caused by muscle-secreted myokine interleukin (IL)-6. This occurrence results in increased AMP-activated protein kinase (AMPK) phosphorylation in adipose tissue, which subsequently activates peroxisome proliferator-activated receptor gamma coactivator 1α and the conversion of white adipocytes to beige in pigs. Therefore, we concluded that MSTN deficiency leads to increased IL-6 secretion in skeletal muscle and activates AMPK in adipocytes, thereby increasing the beige adipose tissue in MSTN-/- pigs.

Sialylated O-glycans and sulfated tyrosines in the NH2-terminal domain of CC chemokine receptor 5 contribute to high affinity binding of chemokines.

The chemokine receptor CCR5 plays an important role in leukocyte chemotaxis and activation, and also acts as a coreceptor for human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV). We provide evidence that CCR5 is O-glycosylated on serine 6 in the NH2 terminus. The O-linked glycans, particularly sialic acid moieties, significantly contribute to binding of the chemokine ligands. By contrast, removal of O-linked oligosaccharide exerted little effect on HIV-1 infection. Sulfation of specific tyrosine residues in the CCR5 NH2 terminus was important for efficient beta-chemokine binding. Thus, as has been observed for the binding of selectins and their ligands, O-linked carbohydrates and tyrosine sulfates play major roles in promoting the interaction of chemokines with CCR5. The resulting flexible arrays of negative charges on the CCR5 surface may allow specific, high-affinity interactions with diverse chemokine ligands. Although this is the first example of O-linked oligosaccharides and tyrosine sulfates playing a role in chemokine binding, the high density of serines, threonines and tyrosines in the N-termini of many CC chemokine receptors suggests that these posttranslational modifications may commonly contribute to chemokine binding.

Sulfated tyrosines contribute to the formation of the C5a docking site of the human C5a anaphylatoxin receptor.

The complement anaphylatoxin C5a and its seven-transmembrane segment (7TMS) receptor play an important role in host defense and in a number of inflammation-associated pathologies. The NH(2)-terminal domain of the C5a receptor (C5aR/CD88) contributes substantially to its ability to bind C5a. Here we show that the tyrosines at positions 11 and 14 of the C5aR are posttranslationally modified by the addition of sulfate groups. The sulfate moieties of each of these tyrosines are critical to the ability of the C5aR to bind C5a and to mobilize calcium. A C5aR variant lacking these sulfate moieties efficiently mobilized calcium in response to a small peptide agonist, but not to C5a, consistent with a two-site model of ligand association in which the tyrosine-sulfated region of the C5aR mediates the initial docking interaction. A peptide based on the NH(2) terminus of the C5aR and sulfated at these two tyrosines, but not its unsulfated analogue or a doubly sulfated control peptide, partially inhibited C5a association with its receptor. These observations clarify structural and mutagenic studies of the C5a/C5aR association and suggest that related 7TMS receptors are also modified by functionally important sulfate groups on their NH(2)-terminal tyrosines.

CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro.

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection.

Clinical isolates of primate immunodeficiency viruses, including human immunodeficiency virus type 1 (HIV-1), enter target cells by sequential binding to CD4 and the chemokine receptor CCR5, a member of the seven-transmembrane receptor family. HIV-1 variants which use additional chemokine receptors are present in the central nervous system or emerge during the course of infection. Simian immunodeficiency viruses (SIV) have been shown to use CCR5 as a coreceptor, but no other receptors for these viruses have been identified. Here we show that two orphan seven-transmembrane segment receptors, gpr1 and gpr15, serve as coreceptors for SIV, and are expressed in human alveolar macrophages. The more efficient of these, gpr15, is also expressed in human CD4(+) T lymphocytes and activated rhesus macaque peripheral blood mononuclear cells. The gpr15 and gpr1 proteins lack several hallmarks of chemokine receptors, but share with CCR5 an amino-terminal motif rich in tyrosine residues. These results underscore the potential diversity of seven-transmembrane segment receptors used as entry cofactors by primate immunodeficiency viruses, and may contribute to an understanding of viral variation and pathogenesis.

CD4-independent binding of SIV gp120 to rhesus CCR5.

CCR5 and CD4 are coreceptors for immunodeficiency virus entry into target cells. The gp120 envelope glycoprotein from human immunodeficiency virus strain HIV-1(YU2) bound human CCR5 (CCR5hu) or rhesus macaque CCR5 (CCR5rh) only in the presence of CD4. The gp120 from simian immunodeficiency virus strain SIVmac239 bound CCR5rh without CD4, but CCR5hu remained CD4-dependent. The CD4-independent binding of SIVmac239 gp120 depended on a single amino acid, Asp13, in the CCR5rh amino-terminus. Thus, CCR5-binding moieties on the immunodeficiency virus envelope glycoprotein can be generated by interaction with CD4 or by direct interaction with the CCR5 amino-terminus. These results may have implications for the evolution of receptor use among lentiviruses as well as utility in the development of effective intervention.

Molecular variation in pigmentation genes contributing to coat colour in native Korean Hanwoo cattle.

Pigmentation genes such as TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (previously TYRP2, or tyrosinase-related protein 2), ASIP (agouti) and MC1R (melanocortin receptor 1) play a major role in cattle coat colour. To understand the genotypic profile underlying coat colour in native Korean Hanwoo cattle and Angus black cattle, portions of the above-mentioned genes were amplified. Sequence analysis revealed variation in the TYRP1 (exon 5) and MC1R genes. Restriction enzyme analysis of these two genes could distinguish between different colours of Hanwoo cattle. Quantitative estimates of melanin and eumelanin in hair from three different-coloured Hanwoo phenotypes and Angus black showed significant differences at the breed and phenotypic levels. Finally, sequence variants in MC1R were associated with total melanin and eumelanin in breeds as well as in Hanwoo phenotypes.

Development of LiF:Mg,Cu,Si TL material (new KLT-300) with a low-residual signal and high-thermal stability.

LiF-based thermoluminescence (TL) materials have been widely used for radiation dosimetry due to their attractive features. LiF:Mg,Cu,P is one of the most sensitive tissue-equivalent TL materials, approximately 40 times more sensitive than LiF:Mg,Ti (TLD-100), but it has two main drawbacks: a thermal loss of the TL sensitivity when annealed at temperatures>240 degrees C, and a relatively high-residual signal. Recently, LiF:Mg,Cu,Na,Si TL material was developed to overcome these drawbacks at the Korea Atomic Energy Research Institute, but it provided only marginal improvements in reducing the residual signal. The newly developed LiF:Mg,Cu,Si TL material has a significantly lower residual signal and a better stability to thermal treatments. In this article, the preparation method and some dosimetric properties (sensitivity and residual signal) of the new LiF:Mg,Cu,Si TL material are presented. At the end of the preparation procedures, a dual-step annealing method is introduced and this has proved as a very efficient method to reduce the high-temperature peak and is the cause of residual signal. Therefore, the high-temperature peak in the glow curve was significantly reduced. The sensitivity is approximately 20 times higher than that of TLD-100 and the residual signal was estimated to be approximately 0.04%.

Histone deacetylase inhibitor M344 significantly improves nuclear reprogramming, blastocyst quality, and in vitro developmental capacity of cloned pig embryos.

M344 is a novel histone deacetylase inhibitor. There is no report on the effect of M344 treatment on the development of pig embryos after somatic cell nuclear transfer (SCNT). In the present study, we investigated the effect of M344 on the blastocyst formation rate in cloned embryos, acetylation level of histone H4 lysine 12 (AcH4K12), and the expression of pluripotency-related genes , , and . Our results indicated that treatment with 5 µ M344 for 6 h improved the development of porcine embryos, in comparison with the untreated group (25.1% ± 5.0 vs. 10.9% ± 2.4; < 0.05). Moreover, M344-treated embryos had increased average fluorescence intensity of AcH4K12 at the pseudo-pronuclear stage ( < 0.05). However, no differences exist in Oct4, NANOG, and SOX2 expression in M344-treated and untreated SCNT blastocysts. In evaluating the effect of M344 on in vivo development, 845 M344-treated embryos were transferred into 3 surrogates, 1 of whom became pregnant and developed 3 fetuses. These findings suggested that M344 elevated the level of histone acetylation, facilitated the nuclear programming, and subsequently improved the developmental competence of pig SCNT embryos.

PO-43 - Differential coagulation factor expression in neuroendocrine prostate cancer (PC), metastatic castrate-resistant PC, and localized prostatic adenocarcinoma.

INTRODUCTION: Neuroendocrine prostate cancer (NEPC) is an aggressive late-stage variant of PC that is often androgen-receptor negative. Most clinicians believe the VTE rate with NEPC is higher than with standard metastatic castration-resistant PC (mCRPC), but NEPC tends to present with bulkier visceral disease and include platinum chemotherapy unlike standard PC. In many solid tumors, a more aggressive phenotype correlates with increased VTE risk and elevated expression of coagulation factors. We previously reported on the differential expression of thrombin and tissue factor (TF) in NEPC versus localized PC and benign prostate tissue with a small NEPC cohort (N=7), which showed overexpression of prothrombin and reduced expression of TF in NEPC. AIM: To compare the expression of coagulation factors of NEPC vs mCRPC (and localized PC control) in an expanded datase. MATERIALS AND METHODS: Fresh frozen tissue biopsies were collected and separated into three cohorts based on pathology: localized PC (N=68), standard mCRPC (N=32), and NEPC (N=21). RNA was isolated and next generation paired-end mRNA sequencing was performed on Illumina Sequencers. F2 Prothrombin (F2), tissue factor (F3), carboxypeptidase (CPB2), fibrinogen (FGG, FGA), PAR-1 (F2R), and PAR-2 (F2RL1) were compared by Wilcoxon tests. RESULTS: Prothrombin had significantly higher expression in NEPC versus standard mCRPC (p <0.001). NEPC trended towards higher expression of CPB2 (p=0.1) and lower expression of F3 (p=0.23) and F2RL1 (p=0.14) compared to mCRPC. Compared to localized PC, both types of advanced disease (NEPC and mCRPC) overexpressed F2, FGA, FGB, and CPB2 (p<0.001) and had decreased expression of F3 and F2RL1 (p <0.001). CONCLUSIONS: Prothrombin is reliably overexpressed in NEPC vs mCRPC and localized PC. Advanced disease (regardless of subtype) is associated with significantly higher expression of prothrombin, fibrinogen, and carboxypeptidase and lower expression of TF and PAR-2. It is possible that there may be PC-specific differences with aggressive disease associated with the thrombin axis vs the more common TF/PAR2 axis commonly seen in other advanced solid tumors. Further research is required to understand these differences in biology and resulting thrombotic and hemostatic outcomes.

Simultaneous resonant x-ray diffraction measurement of polarization inversion and lattice strain in polycrystalline ferroelectrics.

Structure-property relationships in ferroelectrics extend over several length scales from the individual unit cell to the macroscopic device, and with dynamics spanning a broad temporal domain. Characterizing the multi-scale structural origin of electric field-induced polarization reversal and strain in ferroelectrics is an ongoing challenge that so far has obscured its fundamental behaviour. By utilizing small intensity differences between Friedel pairs due to resonant scattering, we demonstrate a time-resolved X-ray diffraction technique for directly and simultaneously measuring both lattice strain and, for the first time, polarization reversal during in-situ electrical perturbation. This technique is demonstrated for BaTiO3-BiZn0.5Ti0.5O3 (BT-BZT) polycrystalline ferroelectrics, a prototypical lead-free piezoelectric with an ambiguous switching mechanism. This combines the benefits of spectroscopic and diffraction-based measurements into a single and robust technique with time resolution down to the ns scale, opening a new door to in-situ structure-property characterization that probes the full extent of the ferroelectric behaviour.

Permeation properties of inward-rectifier potassium channels and their molecular determinants.

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) >> Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.

Permeation and gating of an inwardly rectifying potassium channel. Evidence for a variable energy well.

Permeation, gating, and their interrelationship in an inwardly rectifying potassium (K+) channel, ROMK2, were studied using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode. The gating kinetics of ROMK2 were well described by a model having one open and two closed states. One closed state was short lived (approximately 1 ms) and the other was longer lived (approximately 40 ms) and less frequent (approximately 1%). The long closed state was abolished by EDTA, suggesting that it was due to block by divalent cations. These closures exhibit a biphasic voltage dependence, implying that the divalent blockers can permeate the channel. The short closures had a similar biphasic voltage dependence, suggesting that they could be due to block by monovalent, permeating cations. The rate of entering the short closed state varied with the K+ concentration and was proportional to current amplitude, suggesting that permeating K+ ions may be related to the short closures. To explain the results, we propose a variable intrapore energy well model in which a shallow well may change into a deep one, resulting in a normally permeant K+ ion becoming a blocker of its own channel.

Comparative studies of ribulose-1,5-biphosphate carboxylase/oxygenase from Alcaligenes eutrophus H16 cells, in the active and CABP-inhibited forms.

RuBisCO (D-ribulose-1,5-biphosphate carboxylase/oxygenase; EC 4.1.1.39) has been isolated from the autotrophic hydrogen-oxidizing bacterium Alcaligenes eutrophus H16. Combining photon correlation and sedimentation analysis transport parameters of the enzyme were investigated in the active, (E.CO2.Mg2+) as a ternary complex, and inactive state, (E.CO2.Mg2+.CABP) as a quaternary complex, where RuBisCO is complexed with the transition state analogue CABP (2-C-carboxy-D-arabinitol-1,5-biphosphate). Within experimental error, no difference has been detected between the diffusion and sedimentation coefficients (D020,w = 2.72(+/- 0.07) x 10(-7) cm2 s-1, s020,w = 17.8(+/- 0.5)S) of active and CABP-complexed enzyme thus leading to the conclusion that the molecule, at least in solution, does not assume a different conformation when complexed with CABP.

Crystallization of the activated ternary complex of ribulose-1,5-bisphosphate carboxylase-oxygenase isolated from Rhodospirillum rubrum and from an Escherichia coli clone.

Ribulose-1,5-bisphosphate carboxylase-oxygenase was purified from the photosynthetic bacterium Rhodospirillum rubrum as well as from an Escherichia coli clone overproducing the enzyme. Although the latter enzyme contains 25 additional amino acid residues at the N terminus, both preparations yielded isomorphous tetragonal, bipyramidal crystals of the ternary complex of the enzyme with CO2 and Mg2+. Crystallization is sensitive to variation in pH and to the addition of the transition state analog, 2-carboxyarabinitol-1,5-bisphosphate. The systematic absences in the X-ray diffraction photographs suggest a tetragonal space group P4(3)2(1)2 or the enantiomorph P4(1)2(1)2 with cell dimensions a = b = 83 A, c = 290 A. There is one molecule per asymmetric unit. The resolution on still photographs is 3 A. The crystals are comparable to some of those already published but differ from others.