[Clinical characteristics and correlative factors of dry eye disease associated with graft-versus-host disease].

Objective: To investigate the clinical features of dry eye disease in patients with graft-versus-host disease (GVHD) and to identify the correlative factors that contribute to its severity. Methods: It was a retrospective case series study. A total of 62 patients with dry eye disease caused by GVHD after allogeneic hematopoietic stem cell transplantation (HSCT) were recruited from the First Affiliated Hospital of Soochow University between 2012 and 2020. The study population comprised 38 males (61%) and 24 females (39%), with an average age of (35.29±11.75) years. Only the right eye of each patient was evaluated. The patients were divided into two groups based on the severity of corneal epitheliopathy: a mild group (15 eyes) and a severe group (47 eyes). Demographic information, including gender, age, primary disease, type of allogeneic HSCT, donor-to-recipient information, source of hematopoietic stem cells, systemic GVHD, and the time from HSCT to the first visit, was collected. Ophthalmologic assessments, including the Schirmer Ⅰ test, tear breakup time, corneal epithelial staining, and eye margin assessment, were performed during the first visit to the ophthalmology department and compared between the two groups. Results: The average time from HSCT to the first visit to the ophthalmology department among the 62 patients was (20.26±13.09) months. The median corneal fluorescein staining score was 4.5 points. In the mild group, the main characteristic of corneal staining was scattered punctate staining in the peripheral region in 80% of cases, while in the severe group, corneal staining fused into clumps in both the peripheral region (64%) and the pupillary zone (28%). Results of the Schirmer Ⅰ test were significantly lower in the severe group compared to the mild group (P<0.05). The median total eyelid margin score in the severe group was higher than that in the mild group [9 (7, 12) points vs. 6 (5, 8) points] (P<0.05). The median eyelid congestion score in the severe group was, also higher than that in the mild group [2 (1, 3) points vs. 1 (0, 2) points] (P<0.05). The compatibility between the blood types of the donor and recipient was found to be statistically significant (P<0.05). There was no significant difference in gender, age, family relationship, human leukocyte antigen matching, gender consistency, source of hematopoietic stem cells, or the occurrence of systemic GVHD between the two groups (P>0.05). Conclusions: Patients in the mild group had scattered punctate corneal staining in the peripheral region, while those in the severe group showed fusion of corneal staining into clumps in both the peripheral and pupillary zones. The severity of dry eye disease caused by GVHD was strongly correlated with eyelid margin lesions. A higher degree of eyelid margin lesions indicated more severe dry eye disease caused by GVHD. Additionally, compatibility between the blood types of the donor and recipient may play a role in the development of GVHD-associated dry eye.

l-citrulline prevents asymmetric dimethylarginine-mediated reductions in nitric oxide and nitrosative stress in primary human airway epithelial cells.

BACKGROUND: Asthma is associated with reduced systemic levels of l-arginine and increased asymmetric dimethylarginine (ADMA). This imbalance leads to nitric oxide synthase (NOS) uncoupling with reduced nitric oxide (NO) formation and greater oxidative and nitrosative stress. Whether this imbalance also occurs in bronchial epitheliumof asthmatics is unknown. OBJECTIVES: We used primary human bronchial epithelial cells (HBECs) from asthmatics and healthy controls to evaluate: (i) ADMA-mediated NOS uncoupling reduces epithelial production of NO and increases oxygen and nitrogen reactive species, and (ii) l-citrulline can reverse this mechanism by recoupling NOS, restoring NO production and reducing oxidative and nitrosative stress. RESULTS: In HBECsIL-13 and INFγ stimulated NOS2 and increased NOx levels. The addition of ADMA reduced NOx and increased H2 O2 levels (p<0.001). Treatment with l-citrulline (800, 1600 µm) rescued NOx when the l-arginine media concentration was 25 µm but failed to do so with higher concentrations (100 µm). Under reduced l-arginine media conditions, HBECs treated with l-citrulline increased the levels of argininosuccinate, an enzyme that metabolizes l-citrulline to l-arginine. l-citrulline prevented the ADMA-mediated increase in nitrotyrosine in HBECs in cells from asthmatics and controls. CONCLUSIONS AND CLINICAL RELEVANCE: Increasing ADMA reduces NO formation and increases oxidative and nitrosative stress in airway epithelial cells. l-citrulline supplementation restores NO formation, while preventing nitrosative stress. These results, suggest that l-citrulline supplementation may indeed be a powerful approach to restore airway NO production and may have a therapeutic potential in diseases in which there is a defective production of NO.

Novel inactivating mutation of the calcium-sensing receptor in a young woman with mild hypercalcaemia.

A young woman with mild hypercalcaemia and an inappropriately normal serum parathyroid hormone had parathyroid scintigraphy suggestive of an active ectopic parathyroid tissue in the superior mediastinum. Urinary calcium to creatinine clearance ratio was low, and a subsequent genetic analysis confirmed a novel mutation (Q164K) in the calcium sensing receptor gene, consistent with familial hypocalciuric hypercalcaemia. We propose that this mutation accounts for her clinical and investigational findings, although a double pathology of Q164K and an ectopic parathyroid adenoma is also conceivable.

Tissue-resident alveolar macrophages reduce O3-induced inflammation via MerTK mediated efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3) - one of the six criteria air pollutants - is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung and their number increases following O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. Here, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage tracing experiments showed that 12, 24, and 72 h after exposure to O3 (2 ppm) for 3h all AMØs were tissue-resident origin. Similarly, in humans exposed to FA and O3 (200 ppb) for 135 minutes, we did not observe ~21h post-exposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØ demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK - a key receptor involved in efferocytosis - also resulted in impaired clearance of apoptotic neutrophils followed O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

[Eltrombopag for thrombocytopenia in 24 children after hematopoietic stem cell transplantation].

Objective: To evaluate the efficacy and safety of eltrombopag for children with thrombocytopenia after hematopoietic stem cell transplantation (HSCT). Methods: Clinical data of 24 patients with thrombocytopenia after HSCT,who were treated with eltrombopag in the Department of Pediatrics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University from August 1, 2018 to April 1, 2019 were analyzed retrospectively. The response rate and adverse reactions of eltrombopag were evaluated. Patients were divided into groups by source of hematopoietic stem cells (umbilical cord blood group and peripheral stem cell group) and type of disease (malignant and non-malignant disease group) and the clinical outcomes between groups were compared. Rank Sum test was used for comparisons between groups. Results: Among 24 cases, 15 were males and 9 females, the age of starting eltrombopag was 7.7 (2.6-13.7) years, the time of eltrombopag treatment after HSCT was 27.5 (8.0-125.0) days, the time from treatment to complete response (CR) was 23.5 (6.0-83.0) days, with the treatment course 36.5 (8.0-90.0) days. The total dose of eltrombopag was 1 400(200-5 900) mg. Complete response rate was 92% (22/24),without eltrombopag related adverse reactions. Comparing with peripheral stem cell group (n=8), the course and total dose of eltrombopag in umbilical cord blood group (n=16) were significantly reduced(24.5 (8.0-81.0) vs. 65.5 (35.0-90.0) d, Z=-3.004, P=0.002; 900.0 (200.0-3 850.0) vs. 2 862.5 (1 175.0-5 900.0) mg, Z=-2.604, P=0.007), but no significant differences were found in the time from treatment to complete response, platelet count after 2 weeks of eltrombopag withdrawal or platelet count at the end point of follow-up (all P>0.05). Comparing malignant patients (n=12) and non-malignant patients (n=12), no significant differences were found in the time from treatment to complete response, course, total dose, platelet count after 2 weeks of eltrombopag withdrawal, and platelet count at the end point of follow-up in non-malignant patients (all P>0.05). Conclusion: Eltrombopag is safe and maybe effective for thrombocytopenia after HSCT, especially for umbilical cord blood transplantation.

Pharmacogenetics of medication-related osteonecrosis of the jaw: a systematic review and meta-analysis.

Medication-related osteonecrosis of the jaw (MRONJ) is a severe complication that can develop in patients treated with anti-resorptive drugs. Although the pathogenesis of MRONJ is still unclear, genetic factors have a demonstrated important role. Thus, the aim of this study was to perform a systematic review on the pharmacogenetics of MRONJ. Studies published until March 2019 were retrieved from eight databases and were selected by two independent reviewers. Evidence on several genetic polymorphisms was summarized and a meta-analysis was conducted when possible. Fourteen studies involving 1515 participants were eligible for systematic review. For CYP2C8 rs1934951, no significant difference was observed between the MRONJ and non-MRONJ groups (odds ratio (OR) 2.04, 95% confidence interval (CI) 0.88-4.73, P=0.09). However, a subgroup analysis based on only multiple myeloma status showed a positive association (OR 3.64, 95% CI 1.29-10.30, P=0.01). PPARG rs1152003 was not differently distributed between groups (OR 0.25, 95% CI 0.01-9.92, P=0.46). Also, VEGF rs3025039 was found to be correlated with the occurrence of MRONJ (OR 0.35, 95% CI 0.15-0.82, P=0.02). CYP2C8 rs1934951 (in multiple myeloma patients) and VEGF rs3025039 are associated with the development of MRONJ in patients treated with bisphosphonates. The results are promising and call for new trials with a larger sample to further explore this growing field.

MicroRNA-9501 inhibits breast cancer proliferation and metastasis through regulating Wnt/ß-catenin pathway.

OBJECTIVE: This research was designed to explore the expression characteristics of microRNA-9501 in breast cancer (BCa), and to further explore whether it can influence the development of BCa through the regulation of Wnt/ß-Catenin pathway. PATIENTS AND METHODS: QPCR was carried out to examine microRNA-9501 level in tumor tissue samples and paracancerous ones collected from 42 BCa patients, and the interplay between microRNA-9501 expression and the clinical indicators, as well as the prognosis of BCa patients were analyzed. In addition, we detected microRNA-9501 expression in BCa cell lines by qPCR. Subsequently, microRNA-9501 overexpression model was constructed in BCa cell lines MCF-7 and MDA-MB-231. Then, CCK-8, EdU, cell wound healing, as well as transwell assays, were carried out to evaluate the impact of microRNA-9501 on the biological functions of BCa cells. Finally, the Dual-Luciferase reporting test and tumor formation experiment in nude mice were conducted to further clarify the potential molecular mechanism. RESULTS: QPCR results indicated that microRNA-9501 level in tumor tissue specimens of BCa patients was remarkably higher than that in adjacent ones, and the difference was statistically significant. Compared with patients with high expression of microRNA-9501, patients with lowly-expressed microRNA-9501 had higher tumor stage, higher incidence of lymph node or distant metastasis, and lower overall survival rate. In addition, compared with control group, cells in microRNA-9501 overexpression group showed a significant decrease in proliferation rate, invasiveness, and migration ability. Meanwhile, luciferase reporting assay revealed that overexpression of ß-Catenin remarkably attenuated the luciferase activity of the vector containing wild-type microRNA-9501 sequences, further demonstrating that microRNA-9501 can be targeted by ß-Catenin. Meanwhile, qPCR revealed a negative association between ß-Catenin and microRNA-9501 in BCa tissues. Finally, tumor-bearing experiments in nude mice also demonstrated that microRNA-9501 may suppress the malignant growth of breast tumor. CONCLUSIONS: MicroRNA-9501 expression was found remarkably decreased in BCa tissues and cell lines, which was closely relevant to the pathological stage, metastasis incidence, and prognosis of BCa patients. In addition, microRNA-9501 may suppress the malignant progression of BCa via modulating Wnt/ß-Catenin path-way.

An Fe2IVO2 diamond core structure for the key intermediate Q of methane monooxygenase.

A new paradigm for oxygen activation is required for enzymes such as methane monooxygenase (MMO), for which catalysis depends on a nonheme diiron center instead of the more familiar Fe-porphyrin cofactor. On the basis of precedents from synthetic diiron complexes, a high-valent Fe2(micro-O)2 diamond core has been proposed as the key oxidizing species for MMO and other nonheme diiron enzymes such as ribonucleotide reductase and fatty acid desaturase. The presence of a single short Fe-O bond (1.77 angstroms) per Fe atom and an Fe-Fe distance of 2.46 angstroms in MMO reaction intermediate Q, obtained from extended x-ray absorption fine structure and Mössbauer analysis, provides spectroscopic evidence that the diiron center in Q has an Fe2IVO2 diamond core.

Reversible cleavage and formation of the dioxygen O-O bond within a dicopper complex.

A key step in dioxygen evolution during photosynthesis is the oxidative generation of the O-O bond from water by a manganese cluster consisting of M2(mu-O)2 units (where M is manganese). The reverse reaction, reductive cleavage of the dioxygen O-O bond, is performed at a variety of dicopper and di-iron active sites in enzymes that catalyze important organic oxidations. Both processes can be envisioned to involve the interconversion of dimetal-dioxygen adducts, M2(O2), and isomers having M2(mu-O)2 cores. The viability of this notion has been demonstrated by the identification of an equilibrium between synthetic complexes having [Cu2(mu-eta2:eta2-O2)]2+ and [Cu2(mu-O)2]2+ cores through kinetic, spectroscopic, and crystallographic studies.

Validation of spirometry prediction equations in western Kenya.

SETTING: Community of Eldoret, Kenya. OBJECTIVE: To test the performance of three commonly used spirometry prediction equations in a healthy Kenyan population. DESIGN: Cross-sectional assessment of healthy adults in Eldoret. RESULTS: Of the 331 subjects enrolled in the study, 282 subjects aged 18-85 years (45% males, 55% females) produced high-quality spirograms. Lung function predictions were made using the Global Lung Initiative 2012 (GLI 2012) prediction equations for African Americans, the National Health and Nutrition Examination Survey III (NHANES III) prediction equations for African Americans, and the Crapo prediction equation. Bland-Altman analyses were performed to measure the agreement between observed and predicted spirometry parameters. Overall, the GLI 2012 and NHANES equations for African Americans performed similarly for forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1), significantly overestimating FVC while accurately predicting observed FEV1 values. CONCLUSION: The study brings into question the utility of three major spirometry prediction equations in a Kenyan population. The significant overestimation of FVC by the best-performing equations despite accurate prediction of FEV1 suggests poor performance of these equations in our population.

[Effect of hydrocinnamoyl-L-valyl pyrrolidine on healing quality of deep partial-thickness scald wound in mice].

Objective: To observe the effect of Toll interleukin-1 recptor homology/BB-loop mimetic hydrocinnamoyl-L-valyl pyrrolidine (AS-1) on the healing quality of deep partial-thickness scald wound in mice. Methods: Forty-two adult C57BL/6 mice were divided into sham injury group (SI), scald group (S), early AS-1 treatment group (EAT), early dimethyl sulfoxide (DMSO) control group (EDC), late AS-1 treatment group (LAT), late DMSO control group (LDC) according to the random number table, with 7 mice in each group. Mice in group SI were sham injured without other treatment. Deep partial-thickness scald model with 10% total body surface area was reproduced on the back of the other mice, and the wound was treated by daily wound cleaning with saline and dressing changing with vaseline gauze after injury. Mice in group EAT and those in group LAT were intraperitoneally injected with 20 mg/mL AS-1 50 mg/kg each day respectively from post scald hour (PSH) 8 and post scald day (PSD) 15 on. Mice in group EDC and those in group LDC were intraperitoneally injected with 20 mg/mL DMSO 50 mg/kg each day respectively from PSH 8 and PSD 15 on. On PSD 21, the gross condition of wound healing of mice with scald was observed, and the wound healing rate was calculated. Tissue samples of healed wound were collected and stained with HE and Masson respectively to observe the histomorphological change and fibrosis of collagen, and the percentage of fibrosis of collagen was calculated. The mRNA expressions of interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), transforming growth factor ß1 (TGF-ß1), matrix metalloproteinase-1 (MMP-1), tissue inhibitors of metalloproteinase 1 (TIMP-1), connective tissue growth factor (CTGF), type Ⅰ collagen and type Ⅲ collagen in healed wound tissue were detected by real time fluorescent quantitive reverse transcription polymerase chain reaction. The protein expressions of type Ⅰ collagen and type Ⅲ collagen in healed wound tissue were detected by Western blotting. Skin tissue of mice in group SI at the same area as that observed and collected in mice with scald was performed with the same observation and detection as mentioned above at the same time. Data were processed with one-way analysis of variance and Tukey test. Results: On PSD 21, no abnormal appearance was found in skin tissue of mice in group SI. Wounds of mice in group EAT were healed completely without scar formation, while those in the other four groups were not completely healed with scars formed in different degree. The wound healing rate of mice in group EAT was (97±4)%, close to that of group SI (100%, q=1.753, P>0.05), and both of them were obviously higher than those of groups S, EDC, LAT, and LDC [respectively (83±8)%, (87±6)%, (85±9)%, and (85±7)%, with q values from 4.819 to 6.803, P<0.05 or P<0.01]. On PSD 21, no abnormal appearance was found in morphology of skin tissue of mice in group SI. The morphology of healed wound tissue of mice in group EAT was close to that in group SI, with little epidermis hyalinosis and few newly formed collagen fibers arranged orderly. Epidermis hyalinosis in band- or flake-shape and obvious proliferation of collagen fibers arranged disorderly were observed in healed wound tissue of mice in the other four groups. Much infiltration of inflammatory cells was found in group S. The percentage of fibrosis of collagen in healed wound tissue of mice in group EAT was (30±3)%, close to that of group SI [(30±4)%, q=0.159, P>0.05], and both of them were obviously lower than those of groups S, EDC, LAT, and LDC [respectively (86±9)%, (74±5)%, (82±4)%, and (82±7)%, with q values from 12.080 to 15.530, P values below 0.01]. On PSD 21, compared with those of group SI, the mRNA expressions of IL-1ß, TNF-α, TGF-ß1, MMP-1, and CTGF in healed wound tissue of mice in group S, the mRNA expressions of TGF-ß1 in healed wound tissue of mice in groups EDC and LDC, and the mRNA expression of MMP-1 in healed wound tissue of mice in group LAT were significantly increased (with q values from 4.039 to 5.232, P values below 0.05), while the mRNA expression of TIMP-1 in healed wound tissue of mice in group S was significantly decreased (q=4.921, P<0.05). Compared with those of group S, the mRNA expressions of IL-1ß, TNF-α, TGF-ß1, MMP-1, and CTGF in healed wound tissue of mice in group EAT and the mRNA expressions of IL-1ß and CTGF in healed wound tissue of mice in group LAT were significantly decreased (with q values from 4.418 to 6.402, P<0.05 or P<0.01), while the mRNA expressions of TIMP-1 in healed wound tissue of mice in groups EAT and LAT were significantly increased (with q values respectively 3.929 and 8.299, P<0.05 or P<0.01). Compared with those of group SI, the mRNA and protein expressions of type Ⅲ collagen in healed wound tissue of mice in the other groups and the mRNA and protein expressions of type Ⅰ collagen in healed wound tissue of mice in groups S, EDC, LAT, and LDC were significantly increased (with q values from 7.054 to 11.650, P values below 0.01). Compared with those of group EAT, the mRNA and protein expressions of type Ⅰ collagen in healed wound tissue of mice in groups S, EDC, LAT, and LDC were significantly increased (with q values from 5.156 to 7.451, P<0.05 or P<0.01). Conclusions: AS-1 can effectively promote wound healing and reduce fibrosis degree in the early stage of inflammation response after deep partial-thickness scald in mice, which may be related to its effect in decreasing the expression of inflammation related factors IL-1ß and TNF-α and fibrosis related factors TGF-ß1, MMP-1, CTGF, and type Ⅰ collagen.

Protocatechuate 3,4-dioxygenase. Inhibitor studies and mechanistic implications.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa catalyzes the cleavage of 3,4-dihydroxybenzoate (protocatechuate) into beta-carboxy-cis,cis-muconate. The inhibition constants, Ki, of a series of substrate analogues were measured in order to assess the relative importance of the various functional groups on the substrate. Though important for binding, the carboxylate group is not essential for activity. Compounds with para hydroxy groups are better inhibitors than their meta isomers. Our studies of the enzyme-inhibitor complexes indicate that the 4-OH group of the substrate binds to the active-site iron. Taken together, Mössbauer, EPR, and kinetic data suggest a mechanism where substrate reaction with oxygen is preceded by metal activation of substrate.

Mössbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and Mössbauer spectroscopy. Low temperature Mössbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has Mössbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The Mössbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.

Mössbauer studies of cytochrome c' from Rhodospirillum rubrum.

Cytochrome c' from Rhodospirillum rubrum has been investigated in the ferric form with Mössbauer and EPR spectroscopy. In the pH range from 6 to 9.5, three species are observed which belong to two pH-dependent equilibria with pK values near 6 and 8.5. The pK = 6 transition is resolved only with high-field Mössbauer spectroscopy. For the three species we have determined the zero-field splitting parameters and the hyperfine coupling constants. The data were fitted to a spin Hamiltonian which takes into account a weak mixing of excited S = 3/2 states into the sextet ground manifold. The low temperature spectra clearly show that the quadruple coupling constant deltaEQ is positive for ferricytochrome c' and thus in accord with all other high-spin ferric heme proteins.

Oxygen activating nonheme iron enzymes.

The past year has witnessed significant advances in the study of oxygen-activating nonheme iron enzymes. Thirteen crystal structures of substrate and substrate analog complexes of protocatechuate 3, 4-dioxygenase have revealed intimate details about changes at the enzyme active site during catalysis. Crystallographic data have established a 2-His-1-carboxylate facial triad as a structural motif common to a number of mononuclear nonheme iron enzymes, including isopenicillin N synthase, tyrosine hydroxylase and naphthalene dioxygenase. The first metrical data has been obtained for the high valent intermediates Q and X of methane monooxygenase and ribonucleotide reductase, respectively. The number of enzymes thought to have nonheme diiron sites has been expanded to include alkene monooxygenase from Xanthobacter strain Py2 and the membrane bound alkane hydroxylase from Pseudomonas oleovorans (AlkB). Finally, synthetic complexes have successfully mimicked chemistry performed by both mono- and dinuclear nonheme iron enzymes, such as the extradiol-cleaving catechol dioxygenases, lipoxygenase, alkane and alkene monoxygenases and fatty acid desaturases.

Crystallization of catechol-1,2 dioxygenase from Pseudomonas arvilla C-1.

The metalloenzyme catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 consists of three isozymes formed by combinations of two non-identical subunits; alpha alpha, alpha beta and beta beta; with molecular masses of 59,000, 63,000 and 67,000 Da, respectively. The alpha alpha isozyme crystallizes in the orthorhombic space group C222(1) with unit cell dimensions a = 62.7 A, b = 71.5 A, c = 187.1 A. The rectangular plates diffract to 2.6 A resolution. This is the first dioxygenase to be crystallized that uses catechol as a substrate. Comparison of the structure of this enzyme with protocatechuate 3,4-dioxygenase will provide basic information about the mechanisms of subunit association, substrate selectivity, and the origins of metabolic diversity in enzymes.

Anti-intercellular adhesion molecule-1 antibody and intercellular adhesion molecule-1 gene deficiency do not prevent pulmonary neutrophil recruitment in polymicrobial sepsis.

The intercellular adhesion molecule (ICAM)-1 is expressed constitutively in normal lungs and increased in pulmonary inflammation. Whether increased ICAM-1 expression in the lung contributes to neutrophil sequestration during lung inflammation in sepsis is unclear. We tested this hypothesis in mice after systemic sepsis from cecal ligation and puncture (CLP). ICAM-1 expression in mouse CLP lung tissue was found to increase with time. The time course of lung ICAM-1 up-regulation correlated with increases in lung myeloperoxidase (MPO) activity and neutrophil sequestration by light microscopy. The monoclonal IgG2b rat anti-mouse antibody, an anti-ICAM-1 antibody (YN1/1.7), administered intravenously at doses of 3, 10, or 30 mg/kg, however, did not decrease the lung MPO levels compared with nonimmune rat IgG. In support of these findings, lung MPO content in ICAM-1-deficient mice that underwent CLP was significantly higher than similarly treated ICAM-1-sufficient mice. Our results suggest that neutrophil sequestration in the mouse lung after CLP is not dependent on ICAM-1.

Catalytic oxidation with a non-heme iron complex that generates a low-spin Fe(III)OOH intermediate

The antitumor drug bleomycin (BLM) is proposed to act via a low-spin iron(III) hydroperoxide intermediate called "activated bleomycin". To gain more insight into the mechanistic aspects of catalytic oxidation by these intermediates we have studied the reactivity of [(N4Py)Fe(CH3CN)](ClO4)2 (1) (N4Py = N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) with excess H2O2. Under these conditions a transient purple species is generated, [(N4Py)FeOOH]2- (2), which has spectroscopic features and reactivity strongly reminiscent of activated bleomycin. The catalytic oxidation of alkanes such as cyclohexane, cyclooctane, and adamantane by 1 with H2O2 gave the corresponding alcohols and ketones in up to 31% yield. It was concluded, from the O2 sensitivity of the oxidation reactions, the formation of brominated products in the presence of methylene bromide, and the nonstereospecificity of the oxidation of cis- or trans-dimethylcyclohexane, that long-lived alkyl radicals were formed during the oxidations. Oxidation of alkenes did not afford the corresponding epoxides in good yields but resulted instead in allylic oxidation products in the case of cyclohexene, and cleavage of the double bond in the case of styrene. Addition of hydroxyl radical traps, such as benzene and acetone, led to only partial quenching of the reactivity. The kinetic isotope effects for cyclohexanol formation, ranging from 1.5 in acetonitrile to 2.7 in acetone with slow addition of H2O2, suggested the involvement of a more selective oxidizing species in addition to hydroxyl radicals. Monitoring the UV/Vis absorption of 2 during the catalytic reaction showed that 2 was the precursor for the active species. On the basis of these results it is proposed that 2 reacts through homolysis of the O-O bond to afford two reactive radical species: [(N4Py)Fe(IV)O]2+ and *OH. The comparable reactivity of 1 and Fe-BLM raises the possibility that they react through similar mechanistic pathways.