Identification of the cis­molecular neighbours of the immune checkpoint protein B7­H4 in the breast cancer cell­line SK­BR­3 by proteomic proximity labelling.

The immune checkpoint protein B7­H4 plays an important role in the positive as well as the negative regulation of immune T­cell responses. When expressed on cancer cells, B7­H4 inhibits T­cell activity, and numerous types of cancer cells use upregulation of B7­H4 as a survival strategy. Thus, B7­H4 is a potential target for anticancer drug therapy. Unfortunately, the cell biology of this molecule has yet to be fully elucidated. Even basic properties, such as the nature of B7­H4 interactors, are controversial. In particular, the cis­interactors of B7­H4 on cancer cell plasma membranes have not been investigated to date. The present study used a proteomic proximity­labelling assay to investigate the molecular neighbours of B7­H4 on the surface of the human breast cancer cells SK­BR­3. By comparison to a comprehensive proteome analysis of SK­BR­3 cells, the proximity method detected a relatively small number of low abundance plasma membrane proteins highly enriched for proteins known to modulate cell adhesion and immune recognition. It may be inferred that these molecules contribute to the immunosuppressive behaviour that is characteristic of B7­H4 on cancer cells.

Open-label, pilot study examining sequential therapy with oral tacrolimus and topical tacrolimus for severe atopic dermatitis.

BACKGROUND: Systemic treatment options for generalized atopic dermatitis (AD) are limited. To our knowledge, there have been no prospective trials examining the use of oral tacrolimus, a calcineurin inhibitor, in AD. OBJECTIVES: We assessed the safety and efficacy of sequential therapy with oral tacrolimus and topical tacrolimus in the treatment of generalized AD using the Eczema Area and Severity Index and the Physician Global Assessment scores as the primary end points. METHODS: Twelve patients with AD covering at least 50% body surface area were enrolled. Patients in both phases of the study received sequential therapy with oral and topical tacrolimus over a 14-week treatment period. Eczema Area and Severity Index, Physician Global Assessment, and pruritus scores were calculated at each study visit. RESULTS: Patients recorded a 67% improvement in the Eczema Area and Severity Index score, a 45% improvement in the Physician Global Assessment score, and a 69% reduction in the pruritus score. LIMITATIONS: This investigator-initiated, open-label, single-center, proof-of-concept study lacks a large sample size and placebo control group. CONCLUSION: Sequential therapy with oral tacrolimus and topical tacrolimus may be an effective treatment for AD. A large, randomized control study is warranted.

Biological treatment of H(2)S using pellet activated carbon as a carrier of microorganisms in a biofilter.

Biological treatment is an emerging technology for treating off-gases from wastewater treatment plants. The most commonly reported odourous compound in off-gases is hydrogen sulfide (H(2)S), which has a very low odor threshold. This study aims to evaluate the feasibility of using a biological activated carbon as a novel packing material, to achieve a performance-enhanced biofiltration processes in treating H(2)S through an optimum balance and combination of the adsorption capacity with the biodegradation of H(2)S by the bacteria immobilized on the material. The biofilm was mostly developed through culturing the bacteria in the presence of carbon pellets in mineral media. Scanning electron microscopy (SEM) was used to identify the biofilm development on carbon surface. Two identical laboratory scale biofilters, one was operated with biological activated carbon (BAC) and another with virgin carbon without bacteria immobilization. Various concentrations of H(2)S (up to 125 ppmv) were used to determine the optimum column performance. A rapid startup (a few days) was observed for H(2)S removal in the biofilter. At a volumetric loading of 1600 m(3)m(-3)h(-1) (at 87 ppmv H(2)S inlet concentration), elimination capacity of the BAC (181 gH(2)Sm(-3)h(-1)) at removal efficiency (RE) of 94% was achieved. If the inlet concentration was kept at below 30 ppmv, high H(2)S removal (over 99%) was achieved at a gas retention time (GRT) as low as 2s, a value, which is shorter than most previously reported for biofilter operations. The bacteria population in the acidic biofilter demonstrated capacity for removal of H(2)S in a broad pH range (pH 1-7). There are experimental evidences showing that the spent BAC could be re-used as packing material in a biofilter based on BAC. Overall, the results indicated that an unprecedented performance could be achieved by using BAC as the supporting media for H(2)S biofiltration.

High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5'-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase.

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y.) cofactor and R1 is the site of the conversion of nucleoside diphosphates to 2'-deoxynucleoside diphosphates. A mutant in the R1 subunit of Escherichia coli RNR, E441Q, was generated in an effort to define the function of E441 in the nucleotide-reduction process. Cytidine 5'-diphosphate was incubated with E441Q RNR, and the reaction was monitored by using stopped-flow UV-vis spectroscopy and high-frequency (140 GHz) time-domain EPR spectroscopy. These studies revealed loss of the Y. and formation of a disulfide radical anion and present experimental mechanistic insight into the reductive half-reaction catalyzed by RNR. These results support the proposal that the protonated E441 is required for reduction of a 3'-ketodeoxynucleotide by a disulfide radical anion. On the minute time scale, a second radical species was also detected by high-frequency EPR. Its g values suggest that this species may be a 4'-ketyl radical and is not on the normal reduction pathway. These experiments demonstrate that high-field time-domain EPR spectroscopy is a powerful new tool for deconvolution of a mixture of radical species.

Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Identification of dimethylbenzimidazole as the axial ligand.

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.

Thermodynamic and kinetic studies on carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: the importance of entropy in catalysis.

In the catalytic mechanism of nucleotide reduction, ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the homolytic cleavage of the carbon-cobalt bond of adenosylcobalamin (AdoCbl) at a rate approximately 10(11)-fold faster than the uncatalyzed reaction. Model systems have suggested hypotheses for the thermodynamic basis of this reaction, but relevant measurements of the enzymatic reaction have been lacking. To address this question in a system for which the microscopic rate constants can be measured as a function of temperature, we examined the RTPR-catalyzed exchange reaction. RTPR, in the presence of allosteric effector dGTP and in the absence of substrate, catalyzes carbon-cobalt bond homolysis and formation of a thiyl radical from an active-site cysteine in a concerted fashion [Licht, S., Booker, S. , Stubbe, J. (1999) Biochemistry 38, 1221-1233]. Both the kinetics of cob(II)alamin formation and the amounts of cob(II)alamin formed have been studied as a function of AdoCbl concentration and temperature. Analysis of these data has allowed calculation of a DeltaH of 20 kcal/mol, a DeltaS of 70 cal mol-1 K-1, a DeltaH of 46 kcal/mol, and a DeltaS of 96 cal mol-1 K-1 for carbon-cobalt bond homolysis/thiyl radical formation. The results further show that the enzyme perturbs the equilibrium between the reactant (AdoCbl-bound) state and the product (cob(II)alamin/5'-deoxyadenosine (5'-dA)/thiyl radical state, making them approximately equal in energy. The thermodynamic perturbation, in addition to transition-state stabilization, is required for the large rate acceleration observed. Entropic, rather than enthalpic, factors make the largest contribution in both cases.

The function of adenosylcobalamin in the mechanism of ribonucleoside triphosphate reductase from Lactobacillus leichmannii.

Ribonucleoside triphosphate reductase from Lactobacillus leichmannii catalyzes the reduction of nucleotides to deoxynucleotides and uses adenosylcobalamin as a cofactor. A transient protein-based thiyl radical is essential for catalysis. Studies directed toward the elucidation of the function of adenosylcobalamin during catalysis have shown that formation of the thiyl radical and 5'-deoxyadenosine occurs in a concerted fashion with C-Co bond homolysis, that the homolysis is entropically and not enthalpically driven, that the dimethylbenzimidazole moiety of adenosylcobalamin is the axial ligand during catalysis, and that the C-Co bond is reformed after every turnover.

Evaluation of symptom distress in a bone marrow transplant outpatient environment.

OBJECTIVE: To measure patient perceptions of autologous bone marrow transplantation (ABMT)-associated symptoms in the outpatient setting, assess the efficacy of the established antiemetic protocol, evaluate patient satisfaction, and report patient medication compliance. DESIGN: A prospective, descriptive study of patients with breast cancer who were enrolled in an outpatient ABMT program. SETTING: Duke University Autologous Bone Marrow Transplantation Program. METHODS: Patient perceptions of 12 symptoms were measured by the Symptom Distress Scale (SDS) on the day of admission to the hospital, the day of discharge to the outpatient clinic, after bone marrow reinfusion, and before patient release from the clinic. The number of retching and vomiting episodes was recorded by each patient daily. Patient satisfaction was determined by a standardized personal interview conducted prior to discharge. Patient compliance was assessed by a review of patient medication documentation. RESULTS: Twenty-eight patients were enrolled over 5 months. The median SDS scores for each symptom evaluated revealed that anorexia, nausea, fatigue, insomnia, and bowel problems were the most distressing symptoms patients experienced in the outpatient ABMT program. Scores for pain, negative outlook, cough, diminished concentration, and change in appearance indicated only mild distress associated with these variables. The total number of vomiting episodes ranged from 1 to 33 total episodes per patient per outpatient stay. The percentage of patients experiencing a complete antiemetic response ranged from 24% to 48% over the 4 days after chemotherapy but steadily improved thereafter to a peak of 90% 1 week later. Patient satisfaction results showed that patients preferred being out of the hospital and reported their anxiety controlled although most had some problems with the outpatient clinic or medications required. CONCLUSIONS: Loss of appetite, fatigue, and insomnia have been identified as symptoms that are frequently present during the course of the outpatient ABMT program. Mild, intermittent nausea persists in the outpatient setting for up to 9 days after bone marrow transplant despite continuous combination antiemetic therapy. Patient interviews confirmed the belief that patients enjoy being out of the hospital. Medication compliance is more than 90% in this structured outpatient setting.

Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis.

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near, apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M(r) approx. 38,000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

Steroid production by definitive and fetal zones of the human fetal adrenal gland.

This study was performed to assess the relative contributions of the fetal and definitive zones of the human fetal adrenal gland to "corticoid" (cortisol and perhaps other corticosteroids) and dehydroepiandrosterone sulfate (DHAS) production, and the possible regulatory role of ACTH and the fetal pituitary in the secretion of of these steroids. Corticoid and radioimmunoassayable DHAS or total aromatizable androgen secretion by the isolated definitive and fetal zones of the human fetal adrenal gland between 10-20 weeks gestation has been studied in a superfusion system. Different functional capacities of the two zones were seen; corticoids were found to be secreted primarily by the definitive zone, while DHAS was found to be the main secretory product of the fetal zone. Addition of ACTH (250 ng/ml) or fetal pituitary homogenate produced a 2- to 5-fold stimulation of corticoid production by the definitive zone at all gestational ages studied. DHAS secretion by the fetal zone was also stimulated by ACTH. These results indicate that the definitive and fetal zones of the human fetal adrenal gland at midgestation have the capacity to respond to ACTH with increased corticoid or DHAS secretion, respectively.

Role of hCG in regulation of the fetal zone of the human fetal adrenal gland.

It has been suggested that hCG is a trophic hormone for the fetal zone of the human fetal adrenal gland. To test this hypothesis, the isolated fetal zones of adrenals from eight fetuses (12-17-week gestation age) were superfused in the presence or absence of hCG. Dehydroepiandrosterone sulfate (DHAS) was measured in the superfusion effluent. A significant increase in DHAS production was observed in the presence of hCG. DHAS secretion decreased during the first 60 min in the control and experimental superfusions from 83 +/- 10.0 (mean +/- SE) to 71 +/- 8.0, and from 90 +/- 9.0 to 70 +/- 6.0 ng/100 mg/ml, respectively. In the presence of hCG (250 ng/ml), DHAS secretion increased significantly (P less than 0.01) over the controls to 116 +/- 12.0 at 120 min, and remained above the controls thereafter. These results support the hypothesis that hCG is one of the regulators of DHAS production by the human fetal adrenal gland early in gestation. As we found that ACTh stimulated DHAS secretion in a previous study and as there is indirect evidence for a role of ACTH in DHAS regulation late in pregnancy, these observations suggest dual regulation by hCG and ACTH early in pregnancy, and a possible transition to ACTH regulation of the fetal zone of the human fetal adrenal after midgestation.