Insertion of heterometals into the NifEN-associated iron–molybdenum cofactor precursor

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN–MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems.     

Selective adhesion and mineral deposition by osteoblasts on carbon nanofiber patterns

In an effort to develop better orthopedic implants, osteoblast (bone-forming cells) adhesion was determined on microscale patterns (30 microm lines) of carbon nanofibers placed on polymer substrates. Patterns of carbon nanofibers (CNFs) on a model polymer (polycarbonate urethane [PCU]) were developed using an imprinting method that placed CNFs in selected regions. Results showed the selective adhesion and alignment of osteoblasts on CNF patterns placed on PCU. Results also showed greater attraction forces between fibronectin and CNF (compared with PCU) patterns using atomic force microscope force-displacement curves. Because fibronectin is a protein that mediates osteoblast adhesion, these results provide a mechanism of why osteoblast adhesion was directed towards CNF patterns. Lastly, this study showed that the directed osteoblast adhesion on CNF patterns translated to enhanced calcium phosphate mineral deposition along linear patterns of CNFs on PCU. Since CNFs are conductive materials, this study formulated substrates that through electrical stimulation could be used in future investigations to further promote osteoblasts to deposit anisotropic patterns of calcium-containing mineral similar to that observed in long bones.     

Characterization of the secretion efficiency of a plant signal peptide in Bacillus subtilis.

Summary The ability of the Bacillus subtilis secretion machinery to interact with a heterologous signal peptide was studied using a plant (wheat -amylase) signal peptide. The plant signal peptide was capable of mediating secretion of Escherichia coli alkaline phosphatase and B. amyloliquefaciens levansucrase from B. subtilis. This secretion was dependent on the plant signal peptide, as deletion of five amino acids from the hydrophobic core resulted in a block of secretion. Attempts to improve the efficiency of the plant signal peptide in B. subtilis were made by increasing the length of the hydrophobic core from 10 to 16 residues by insertion of 2, 4, 5 or 6 amino acids. None of the alterations improved the secretion efficiency relative to the wild-type plant signal peptide.     

Three new observational scales for use in Dutch nursing homes: scales from the Resident Assessment Instrument for Activities of Daily Living, cognition and depression

The reliability and validity of three MDS scales for ADL, cognition and depression are described. The scales consist of items of the Minimum Data Set of the Resident Assessment Instrument and are available just after an MDS assessment. Data collection took place in nine Dutch nursing homes (N = 227) and consisted of three MDS assessments within one month to determine reliability. Several criterion measures were assessed in order to determine convergent validity. Intra- and inter-rater reliability and internal consistency were determined as well as correlation coefficients of the criterion measures and the MDS scales. All three MDS scales appear reliable, especially the ADL-Hierarchy has very good psychometric properties (intra- and inter-rater Intra Class Correlation were 0.81 and 0.83, respectively). Convergent validity of the ADL-Hierarchy and the Cognitive Performance Scale is good, the Depression Rating Scale appears valid in residents with moderate cognitive disorders at the most, but the results are more difficult to interpret in residents with severe cognitive disorders. The MDS scales appear useful in clinical practice and for research purposes in the Dutch nursing homes.     

Chronic pain in dementia and in disorders with a high risk for congnitive impairment

Ageing increases the risk for the etiology of chronic pain and dementia. hence, the increase in the number of elderly people implies that the number of elderly with dementia suffering from chronic pain will increase as well. A key question relates to if and how patients with dementia perceive pain. the inadequateness of pain assessment, particularly in a more advanced stage, is also reflected in a decreased use of analgesics by elderly people with dementia. Insight into possible changes in pain experience as have been observed in the few available clinical studies, could be enhanced by knowledge about the neuropathology which may differ per subtype of dementia. It is striking that pain has not been examined in degenerative diseases of the central nervous system with a high risk for cognitive impairment such as Parkinson's disease and multiple sclerosis. In these disorders, pain is a prominent clinical symptom and to date it is not known whether the experience of pain will change in a stage in which patients become cognitively impaired. Finally, a number of instruments which are most appropriate to assess pain in communicative and non-communicative patients are discussed.     

Variable-temperature,variable-field magnetic circular dichroism spectroscopic study of NifEN-bound precursor and “FeMoco”

NifEN plays a key role in the biosynthesis of the iron–molybdenum cofactor (FeMoco) of nitrogenase. A scaffold protein that hosts the conversion of a FeMoco precursor to a mature cofactor, NifEN can assume three conformations during the process of FeMoco maturation. One, designated ΔnifB NifEN, contains only two permanent [Fe₄S₄]-like clusters. The second, designated NifEN^Precursor, contains the permanent clusters and a precursor form of FeMoco. The third, designated NifEN^“FeMoco”, contains the permanent [Fe₄S₄]-like clusters and a fully complemented, “FeMoco”-like structure. Here, we report a variable-temperature, variable-field magnetic circular dichroism spectroscopic investigation of the electronic structure of the metal clusters in the three forms of dithionite-reduced NifEN. Our data indicate that the permanent [Fe₄S₄]-like clusters are structurally and electronically conserved in all three NifEN species and exhibit spectral features of classic [Fe₄S₄]⁺ clusters; however, they are present in a mixed spin state with a small contribution from the S > ½ spin state. Our results also suggest that both the precursor and “FeMoco” have a conserved Fe/S electronic structure that is similar to the electronic structure of FeMoco in the MoFe protein, and that the “FeMoco” in NifEN^“FeMoco” exists, predominantly, in an S = 3/2 spin state with spectral parameters identical to those of FeMoco in the MoFe protein. These observations provide strong support to the outcome of our previous EPR and X-ray absorption spectroscopy/extended X-ray absorption fine structure analysis of the three NifEN species while providing significant new insights into the unique electronic properties of the precursor and “FeMoco” in NifEN.     

Sterven met dementie: wat weten wij ervan?

Dying with dementia: what do we know about it? Death with dementia is increasingly common, yet research on end of life with dementia and end-of-life care for such patients has been sparse. This article reviews recent studies in this area, most of which were done in US nursing homes. Research focused on five domains: prognosis, decision making, treatment, patient’s health and suffering, and family’s circumstances and satisfaction with care. Prognostication focused on developing risk scores for mortality within 6 months or a year, and while decision making was usually studied qualitatively, the other three domains were largely covered by a series of small, retrospective studies. Future direction in research is discussed, including the ongoing CASCADE project in Boston and the Dutch End of Life with Dementia Study (DEOLD). Both of these prospective studies in nursing home residents assess decision making, as well as factors associated with family’s satisfaction and patient suffering. These studies will provide insight into interventions that are most likely to improve end of life care of patients with dementia in the respective countries and elsewhere. Tijdschr Gerontol Geriatr 2007; 38: 288-297     

Molecular insights into nitrogenase FeMo cofactor insertion: the role of His 362 of the MoFe protein α subunit in FeMo cofactor incorporation

The assembly of the complex iron-molybdenum cofactor (FeMoco) of nitrogenase molybdenum-iron (MoFe) protein has served as one of the central topics in the field of bioinorganic chemistry for decades. Here we examine the role of a MoFe protein residue (His alpha362) in FeMoco insertion, the final step of FeMoco biosynthesis where FeMoco is incorporated into its binding site in the MoFe protein. Our data from combined metal, activity and electron paramagnetic resonance analyses show that mutations of His alpha362 to small uncharged Ala or negatively charged Asp result in significantly reduced FeMoco accumulation in MoFe protein, indicating that His alpha362 plays a key role in the process of FeMoco insertion. Given the strategic location of His alpha362 at the entry point of the FeMoco insertion funnel, this residue may serve as one of the initial docking points for FeMoco insertion through transient ligand coordination and/or electrostatic interaction.     

Integrating fasciolosis control in the dry cow management: the effect of closantel treatment on milk production

The liver fluke Fasciola hepatica is a parasite of ruminants with a worldwide distribution and an apparent increasing incidence in EU member states. Effective control in dairy cattle is hampered by the lack of flukicides with a zero-withdrawal time for milk, leaving the dry period as the only time that preventive treatment can be applied. Here, we present the results of a blinded, randomized and placebo-controlled trial on 11 dairy herds (402 animals) exposed to F. hepatica to 1) assess the effect of closantel treatment at dry-off (or 80-42 days before calving in first-calving heifers) on milk production parameters and 2) evaluate if a number of easy-to-use animal parameters is related to the milk production response after treatment. Closantel treatment resulted in a noticeable decrease of anti-F. hepatica antibody levels from 3-6 months after treatment onwards, a higher peak production (1.06 kg) and a slightly higher persistence (9%) of the lactation, resulting in a 305-day milk production increase of 303 kg. No effects of anthelmintic treatment were found on the average protein and fat content of the milk. Milk production responses after treatment were poor in meagre animals and clinically relevant higher milk production responses were observed in first-lactation animals and in cows with a high (0.3-0.5 optical density ratio (ODR)), but not a very high (≥0.5 ODR) F. hepatica ELISA result on a milk sample from the previous lactation. We conclude that in dairy herds exposed to F. hepatica, flukicide treatment at dry-off is a useful strategy to reduce levels of exposure and increase milk production in the subsequent lactation. Moreover, the results suggest that treatment approaches that only target selected animals within a herd can be developed based on easy-to-use parameters.