Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving–Williams series (Mn^II < Fe^II < Ni^II < Co^II < Cu^II > Zn^II). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn^II and Fe^II in vitro, thus diverging from the Irving–Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe^II over Mn^II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine–valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.Half of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation. The diiron cofactor of the ferritin-like superfamily of proteins is particularly versatile (2). While ferritin itself simply oxidizes and sequesters iron (3), in other family members the diiron center acts as a transient one- or two-electron oxidant. In the R2 subunits of class I ribonucleotide reductases (RNRs) it generates a redox-active tyrosyl radical (4, 5), whereas in the bacterial multicomponent monooxygenases (BMMs) it catalyzes the hydroxylation of a variety of hydrocarbons (6). For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7–14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn^IV/Fe^III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7, 8). These recent developments highlight the complexity of correctly identifying the metals that make up native metallocofactors. While the metal preferences of some primary coordination motifs are well known and distinct, others are more promiscuous and less well understood. Manganese and iron are two of the most important and versatile metals for biological redox chemistry. Their primary ligand preferences are very similar, and their binding sites in enzymes often appear virtually identical (15). However, their redox potentials differ greatly, and correct discrimination between them is therefore paramount for redox-active enzymes (16). Metal specificity is commonly discussed in terms of the intracellular availability of metal ions and the Irving–Williams series of metal complex stabilities (Mn^II < Fe^II < Ni^II < Co^II < Cu^II > Zn^II) (17). Manganese is generally present in cells at a lower concentration than iron (18) and also has a lower predicted complex stability. The biochemical selection of manganese over iron thus presents an intricate problem. Metallochaperones are required for correct metallation of some metalloproteins (19–22), but to date no chaperones have been identified for manganese, and it is unclear whether they are required for diiron clusters (16). Protein-folding location or general metal status can also control metallation (16, 23). However, these mechanisms cannot be used for proteins with mixed-metal cofactors.The heterodinuclear Mn/Fe cofactor recently discovered in a protein from Mycobacterium tuberculosis provides an ideal model system to study manganese/iron metallation. The protein was found to belong to a novel group of R2-like proteins, denoted R2-like ligand-binding oxidases (R2lox) (11, 24). These proteins are particularly interesting because the primary protein-derived metal-binding ligands are identical in both metal sites, and also identical to the closely related diiron-binding BMMs. To verify that the mixed-metal cofactor is a common feature of the R2lox group, here we characterize a homolog from the thermophilic bacterium Geobacillus kaustophilus (GkR2loxI). Using crystallography, spectroscopy, and quantum chemical calculations, we show that site-specific metal discrimination is inherent to the protein structure and diverges from the Irving–Williams series without requiring auxiliary factors in vitro. The mixed-metal cofactor activates oxygen and catalyzes the formation of an ether cross-link in the protein scaffold, demonstrating the chemical potential of this cofactor.     

Endocrinology: Subcutaneous goserelin versus intranasal buserelin for pituitary down-regulation in patients undergoing IVF: a randomized comparative study

One-hundred women undergoing ovarian stimulation with gonadotrophin-releasinghormone agonist (GnRH-a) and a human menopausal gonadotrophin(HMG) for in-vitro fertilization (IVF) participated in thisrandomized comparative study. The effectiveness of long-actings.c. goserelin (Zoladex depot; 49 patients) and intranasally(i.n.) administrated buserelin acetate (Suprefact; 51 patients)for pituitary down-regulation was compared. Treatment with s.c.goserelin (3.6 mg) or i.n. buserelin acetate (200 µg;6 times/day) was started on day 21–23 of the cycle. Stimulationwith 150 IU of HMG/day was started after at least 11 days ofGnRH-a treatment. There were no differences in the time requiredfor follicular development nor in the clinical outcome betweengroups treated with either goserelin or buserelin. The numberof oocytes recovered in the goserelin group was 6.7 ±5.0 versus 6.3 ± 4.9 in the buserelin group. There were11 pregnancies after the use of goserelin (22.4%) and 12 pregnanciesin those given buserelin (24.0%). The number of HMG ampoulesneeded for follicular maturation was higher in the goserelingroup (27.9 ± 7.8) than in the buserelin group (24.6± 7.8, P < 0.05). The patients given buserelin sufferedsignificantly more from tiredness, depression, headache andabdominal pain than those receiving goserelin, whereas therewere no differences between the groups in experiencing mentalirritability, nausea and swelling. Subcutaneous goserelin depotinjection offers a useful alternative for pituitary down-regulationin IVF stimulation.     

Psychosocial Nursing in Radiotherapy Cancer Wards in Finland

Abstract. In this study the specialized nurses, nurse generalists and practical nurses working in radiotherapy cancer wards in Finland are estimating their capabilities for responding to the psychosocial needs of cancer patients. The methods they use to respond to their patients and the results achieved are also estimated. Details were also elicited of the barriers to the provision of high standard nursing which these people perceived. The length of education correlated significantly with the estimated capabilities of nursing personnel to meet the psychosocial needs of the cancer patients. The practical nurses estimated their capabilities to be in all respects at a lower level than did the nurse generalists and specialized nurses.     

Psychotropic drugs and impairment of psychomotor functions

The present work deals with the effects of psychotropic drug therapy on the operation of psychomotor functions used in a clinical examination of suspected drunken drivers. 100 psychiatric mental, but otherwise healthy, patients were examined; the type of medication and the number of drugs used varied greatly. In 71 cases the mean degree of error in the clinical examination was higher, and, in several of these, markedly higher than the reference values obtained earlier on suspected drunken drivers when the blood contained very small amounts of alcohol or none at all. In 18 cases coarsely-divided nystagmus was registered in patients on psychotropes. This is an obvious sign of a marked side-effect of medication but was present more infrequently than in subjects with after ingestion of alcohol.The present results indicate that application of the clinical examination method, which was originally developed for and related to the examination of alcohol cases, to subjects on psychotropes is adequate, and it is possible with clinical examination to obtain valuable medicolegal information on the impairment of physiological functions. The present review of suspected dragged drivers examined in Helsinki in 1969–1972 also supports this view.This study was supported by grants from the Sigrid Jusélius-Foundation and the Finnish Foundation for Alcohol Studies, Helsinki, Finland.     

Attendance pattern and continuity of dental care of Finnish adults over a 5 year period

The study deals with the frequency of using dental services and continuity of visting the same dentist by adults in Finland during the 5 years up to 1981. A representative sample of 17-65 year old Finnish adults was interviewed by telephone or when this was not possible, personally. The data were collected by the Central Statistical Office of Finland in autumn 1981. Twenty-two per cent of the subjects had not visited a dentist during the past 5 years. One-third had had more than three treatment courses during the same period. The number of treatment periods increased with higher educational and professional status. Half of the subjects who had had more than one treatment period had continued to attend the same dentist during different treatment periods. Young persons had changed dentist more often than older ones. The most common reason for changing dentist was change of residence. Nine percent did not wish to continue treatment with the same dentist. Income, age, profession and region of living accounted for 10% of the number of changes of dentist.     

Notch restricts lymphatic vessel sprouting induced by vascular endothelial growth factor

Notch signaling plays a central role in cell-fate determination, and its role in lateral inhibition in angiogenic sprouting is well established. However, the role of Notch signaling in lymphangiogenesis, the growth of lymphatic vessels, is poorly understood. Here we demonstrate Notch pathway activity in lymphatic endothelial cells (LECs), as well as induction of delta-like ligand 4 (Dll4) and Notch target genes on stimulation with VEGF or VEGF-C. Suppression of Notch signaling by a soluble form of Dll4 (Dll4-Fc) synergized with VEGF in inducing LEC sprouting in 3-dimensional (3D) fibrin gel assays. Expression of Dll4-Fc in adult mouse ears promoted lymphangiogenesis, which was augmented by coexpressing VEGF. Lymphangiogenesis triggered by Notch inhibition was suppressed by a monoclonal VEGFR-2 Ab as well as soluble VEGF and VEGF-C/VEGF-D ligand traps. LECs transduced with Dll4 preferentially adopted the tip cell position over nontransduced cells in 3D sprouting assays, suggesting an analogous role for Dll4/Notch in lymphatic and blood vessel sprouting. These results indicate that the Notch pathway controls lymphatic endothelial quiescence, and explain why LECs are poorly responsive to VEGF compared with VEGF-C. Understanding the role of the Notch pathway in lymphangiogenesis provides further insight for the therapeutic manipulation of the lymphatic vessels.