Enhancing the photoluminescence of surface anchored metal-organic frameworks: mixed linkers and efficient acceptors.

We present two approaches to enhance the photoluminescence quantum yield (PLQY) of surface-anchored metal-organic frameworks (SURMOFs). In the first approach we fabricate SURMOFs from a mix of an emissive linker with an optically-inert linker of equivalent length, diluting the emissive linker while maintaining the SURMOF structure. This approach enhances the internal PLQY. However, the increase in internal PLQY is achieved at the expense of a drastic reduction in optical absorption, thus the external PLQY remains low. To overcome this limitation, a second approach is explored wherein energy-accepting guest chromophores are infiltrated into the framework of the active linker. At the correct acceptor concentration, an internal PLQY of 52% - three times higher than the previous approach - is achieved. Additionally, the absorption remains strong leading to an external PLQY of 8%, an order of magnitude better than the previous approach. Using this strategy, we demonstrate that SURMOFs can achieve PLQYs similar to their precursor chromophores in solution. This is of relevance to SURMOFs as emitter layers in general, and we examine the optimized emitter layer as part of a photon upconversion (UC) SURMOF heterostructure. Surprisingly, the same PLQY is not observed after triplet-triplet annihilation in the UC heterostructure as after its normal photoexcitation (although the UC layers exhibit low thresholds consistent with those reported in our previous work). We discuss the potential bottlenecks in energy transport that could lead to this unexpected reduction in PLQY after excitation via triplet-triplet annihilation, and how future design of SURMOF UC multilayers could overcome these limitations.

Levofloxacin-associated hypoglycaemia complicated by pontine myelinolysis and quadriplegia.

BACKGROUND: Central pontine myelinolysis (CPM) usually presents in chronic alcoholics and in patients in whom hyponatraemia has been corrected rapidly. However, CPM may occur in other clinical circumstances, including patients with severe hypoglycaemia. We describe the occurrence of CPM and quadriplegia in a patient who experienced fluoroquinolone-associated severe hypoglycaemia. CASE REPORT: A 63-year-old man with Type 2 diabetes mellitus was admitted to hospital for resection of a large liposarcoma. Renal-dose levofloxacin was utilized as part of an antimicrobial regimen to treat post-operative peritonitis. On days 6-8 of levofloxacin therapy, the patient experienced recurrent hypoglycaemia despite total parenteral nutrition, 10% dextrose containing fluids and cessation of insulin therapy 3 days prior to the first hypoglycaemic episode. Hypoglycaemia resolved within 24 h of stopping levofloxacin. After a final and severe hypoglycaemic event, the patient developed quadriplegia and tonic left deviation of gaze. Magnetic resonance imaging revealed a high-intensity lesion in the central pons consistent with CPM. CONCLUSIONS: Fluoroquinolones should be considered as a potential cause of hypoglycaemia. Severe hypoglycaemia has the potential to cause white matter lesions in the pons. Putative mechanisms include failure of membrane ion channels, oligodendrocyte apoptosis and oxidative stress of glucose reperfusion. Fluoroquinolone-associated hypoglycaemia and hypoglycaemia-induced quadriplegia are both rare and we believe this is the first case report linking the two events.

Iron assimilation and transcription factor controlled synthesis of riboflavin in plants.

Iron homeostasis is vital for many cellular processes and requires a precise regulation. Several iron efficient plants respond to iron starvation with the excretion of riboflavin and other flavins. Basic helix-loop-helix transcription factors (TF) are involved in the regulation of many developmental processes, including iron assimilation. Here we describe the isolation and characterisation of two Arabidopsis bHLH TF genes, which are strongly induced under iron starvation. Their heterologous ectopic expression causes constitutive, iron starvation independent excretion of riboflavin. The results show that both bHLH TFs represent an essential component of the regulatory pathway connecting iron deficiency perception and riboflavin excretion and might act as integrators of various stress reactions.

Multiplicity of ammonium uptake systems in Corynebacterium glutamicum: role of Amt and AmtB.

In Corynebacterium glutamicum, a Gram-positive soil bacterium widely used in the industrial production of amino acids, two genes encoding (putative) ammonium uptake carriers have been described. The isolation of amt was the first report of the sequence of a gene encoding a bacterial ammonium uptake system combined with the characterization of the corresponding protein. Recently, a second amt gene, amtB, with so far unknown function, was isolated. The isolation of this gene and the suggestion of a new concept for ammonium acquisition prompted the reinvestigation of ammonium transport in C. glutamicum. In this study it is shown that Amt mediates uptake of (methyl)ammonium into the cell with high affinity and strictly depending on the membrane potential. As shown by the determination of K:(m) at different pH values, ammonium/methylammonium, but not ammonia/methylamine, are substrates of Amt. AmtB exclusively accepts ammonium as a transport substrate. In addition, hints of another, until now unknown, low-affinity, ammonium-specific uptake system were found.

Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding.

The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.

AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum.

The uptake and assimilation of nitrogen sources is effectively regulated in bacteria. In the Gram-negative enterobacterium Escherichia coli, the NtrB/C two-component system is responsible for the activation of transcription of different enzymes and transporters, depending on the nitrogen status of the cell. In this study, we investigated regulation of ammonium uptake in Corynebacterium glutamicum, a Gram-positive soil bacterium closely related to Mycobacterium tuberculosis. As shown by Northern blot hybridizations, regulation occurs on the level of transcription upon nitrogen starvation. In contrast to enterobacteria, a repressor protein is involved in regulation, as revealed by measurements of methylammonium uptake and beta-galactosidase activity in reporter strains. The repressor-encoding gene, designated amtR, was isolated and sequenced. Deletion of amtR led to deregulation of transcription of amt coding for the C. glutamicum (methyl)ammonium uptake system. E. coli extracts from amtR-expressing cells were applied in gel retardation experiments, and binding of AmtR to the amt upstream region was observed. By deletion analyses, a target motif for AmtR binding was identified, and binding of purified AmtR protein to this motif, ATCTATAGN1-4ATAG, was shown. Furthermore, the binding of AmtR to this sequence was proven in vivo using a yeast one-hybrid system. Subsequent studies showed that AmtR not only regulates transcription of the amt gene but also of the amtB-glnK-glnD operon encoding an amt paralogue, the signal transduction protein PII and the uridylyltransferase/uridylyl-removing enzyme, key components of the nitrogen regulatory cascade. In summary, regulation of ammonium uptake and assimilation in the high G+C content Gram-positive bacterium C. glutamicum differs significantly from the mechanism found in the low G+C content Gram-positive model organism Bacillus subtilis and from the paradigm of nitrogen control in the Gram-negative enterobacteria.

The role of osteoprogenitors in vascular calcification.

Calcification is a component of vascular disease that usually occurs in concert with atheroma formation but through distinct pathophysiological processes. Vessel wall osteoprogenitor cells known as calcifying vascular cells can form bone matrix proteins and calcified nodules, analogous to osteoblastic differentiation in bone. These cells have been isolated from the tunica media of bovine and human arteries, and both in-vitro tissue culture models and mouse models of vascular calcification have been established. Studies of the effects of diabetes mellitus, hyperlipidemia, estrogens and glucocorticoids on calcifying vascular cell function provide insight into the relationship between common human disease states and vascular calcification.

Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins.

The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PH (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum.

Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I.

The Corynebacterium glutamicum glutamine synthetase I (GSI) structural gene glnA was cloned by a PCR approach using oligonucleotide primers derived from conserved amino acid sequences of the GSI proteins from various bacteria. Disruption or deletion of this gene in C. glutamicum led to a glutamine auxotrophic phenotype and complete loss of glutamine synthetase activity, indicating the key role of this enzyme in nitrogen metabolism. Additionally, indications for a second glutamine synthetase, GSII, were found.

Localization of tolbutamide binding sites on human serum albumin using titration calorimetry and heteronuclear 2-D NMR.

The sulfonylureas are a class of oral hypoglycemic agents used to treat type II diabetes mellitus, and tolbutamide is a "first generation" member of this family. It is a nonpolar, weakly acidic drug that binds to serum albumin in the circulation. In the present study, we have examined the interactions of tolbutamide with human serum albumin by isothermal titration calorimetry and heteronuclear multiple-quantum coherence NMR spectroscopy. Calorimetric titrations revealed that tolbutamide binds to albumin at three independent sites with the same or comparable affinity. This result was independently confirmed by NMR experiments which resolved three resonances at 1H chemical shifts of 2.07, 2.11 and 2.14 ppm, corresponding to [methyl-13C]tolbutamide bound to three discrete binding sites. The binding affinity quantitated by calorimetry (Kd = 21 +/- 9 microM at pH 7.4, 37 degrees C) was approximately 5 times lower than the most frequently reported value. Tolbutamide titrations of albumin complexed with three other drugs whose binding sites have been localized by X-ray crystallography (salicylate, clofibric acid, and triiodobenzoic acid) demonstrated direct competition for common binding sites. NMR experiments with samples containing [methyl-13C]tolbutamide and these competing drugs permitted assignment of the resonances at 2.07 and 2.14 ppm to tolbutamide bound to the aspirin sites in albumin subdomains IIIA and IIA, respectively. These findings permit the first assignment of tolbutamide binding sites to specific locations on the albumin molecule within the context of the recently published crystal structure of human serum albumin. In addition, this information provides a molecular basis for predicting unfavorable drug interactions involving tolbutamide in patients with type II diabetes.

Ligand-protein electrostatic interactions govern the specificity of retinol- and fatty acid-binding proteins.

Cellular retinol-binding protein II (CRBP-II) and intestinal fatty acid-binding protein (I-FABP) are both expressed in small intestinal enterocytes and exhibit 31% sequence identity. I-FABP binds a single molecule of long-chain fatty acid and forms an ion-pair electrostatic interaction between the cationic side chain of arginine-106 and the anionic fatty acid carboxyl group. In contrast, CRBP-II binds all-trans-retinol or -retinal and contains a glutamine residue in the corresponding position, residue 109. We have characterized and compared the interactions of fatty acids and retinoids with I-FABP, CRBP-II, and two reciprocal mutant proteins. The mutants were designated CRBP-II(Q109R), where glutamine-109 was replaced by arginine, and I-FABP(R106Q), where arginine-106 was replaced by glutamine. As monitored by titration calorimetry and carbon-13 NMR spectroscopy, the fatty acid-binding properties of CRBP-II(Q109R) were found to be essentially identical to those of wild-type I-FABP. Both proteins bound 1 molecule of fatty acid with identical affinities (Kd = 0.2 microM). The enthalpic contribution to the total free energy of binding was large for both proteins: 66% and 87%, respectively. In addition, the carboxyl groups of fatty acids bound to both proteins were solvent-inaccessible. There was little or no change in the ionization state of the bound fatty acid over a wide pH range, as monitored by the chemical shift of the fatty acid carboxyl 13C resonance. Furthermore, the binding of fatty acid to both proteins was accompanied by a selective perturbation of the guanidino 13C resonance of a single arginine residue.(ABSTRACT TRUNCATED AT 250 WORDS)