Resonance raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the nu Fe-CO region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the nu Fe-CO modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the "open" configuration and 3.8 in the "closed" conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the "open" conformation. At pH 7, the pocket is found to be "open" approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various nu Fe-CO and nu C-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to nu Fe-CO = 518 cm-1 (nu C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the nu Fe-CO region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the nu Fe-CO modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.     

Resonance Raman studies of iron spin and axial coordination in distal pocket mutants of ferric myoglobin

We have used resonance Raman spectroscopy to study 11 distal pocket mutants and the "wild type" and native ferric sperm whale myoglobin. The characteristic Raman core-size markers v4, v3, v2, and v10 are utilized to assign the spin and coordination state of each sample. It is demonstrated that replacements of the distal and proximal histidines can discriminate against H2O as a sixth ligand and favor a pentacoordinate Fe3+ atom. Soret absorption band blueshifts are correlated with the pentacoordinate heme environment. One E7 replacement (Arg) leads to an iron spin state change and produces a low spin species. The Glu and Ala mutations at position E11 leave the protein's spin and coordination unaltered. A laser-induced photoreduction effect is observed in all pentacoordinate mutants and seems to be correlated with the loss of the heme-bound water molecule.     

Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin

Declining nicotinamide adenine dinucleotide (NAD⁺) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD⁺ levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD⁺ precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD⁺ levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD⁺ levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD⁺ levels in NEVs and decreases NEV levels of Aβ42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin–Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD⁺ levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain.     

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.     

Conformational interconversion in protein crystals.

We present evidence that the structure of carbonmonoxy myoglobin crystals can be altered by lowering the pH. This structural change is monitored by the characteristic Fe-CO Raman modes at 508 and 491 cm-1 and is thought to involve a localized distal pocket transition from a "closed" conformation at pH 7 to a more "open" conformation at pH 4. These changes take place in the crystal without loss of intensity of a conformationally sensitive Raman mode at 252 cm-1 that signals a partial unfolding of the globin structure in solution. Quantitative studies, which monitor the open and closed populations as a function of laser photolysis, demonstrate that the interconversion rates (k+/-) in solution at 298 K are fast compared to the photolysis and CO entry rates (i.e. k+/- much greater than 10(3) s-1), while in frozen samples the interconversion is much slower than the experimental time scale (minutes). Since the open conformation is a minority species at pH 7, rapid exchange in aqueous solution is a necessary condition for this species to play a functional role. In the crystal, the interconversion rates are slowed compared to solution and begin to approach the photolysis rate (i.e. k+/- approximately 10(3) to 10(4) s-1). This indicates that the barriers for conformational exchange are increased in the crystal environment, compared to the solution, apparently due to the packing forces of the surrounding molecules. X-ray and neutron diffraction studies of MbCO crystals at high and low pH are needed to characterize the details of the structural changes and to test the hypothesis that closed and open distal pocket structures are associated with the 508 and 491 cm-1 Fe-CO modes.     

Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography—mass spectrometry

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E₄ (LTE₄). Oxygen-18 labelling of LTE₄ methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE₄ to the extent of 89.8% ([¹⁸O₂]LTE₄) and one oxygen-18 atom to the extent of 9.4% ([¹⁶O¹⁸O]LTE₄), with only 0.7% remaining unchanged ([¹⁶O₂]LTE₄). [¹⁸O₂]LTE₄ was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4°C for up to 20 h. [¹⁸O₂]LTE₄ was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE₄ in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE₄ in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.     

Adenoid cystic carcinoma intermingled with ductal carcinoma of the breast: a case report and review of the literature

ABSTRACT: INTRODUCTION: Adenoid cystic cancer of the breast is a rare condition, and even rarer are the cases where it is histologically mixed with other variants of cancer within a single lesion. In this report, one of the few cases of mixed adenoid cystic breast cancer intermingled with the infiltrating ductal variant is presented. A subsequent review of the relevant literature presents the existing experience in treating mixed breast cancers with adenoid cystic components with regard to diagnosis, treatment, and prognosis. CASE PRESENTATION: We describe a case of mixed adenoid cystic cancer of the breast with infiltrating ductal carcinoma in a 67-year-old Caucasian woman who underwent mastectomy with sentinel node biopsy. CONCLUSION: Surgery remains the cornerstone of treatment of these patients, and radiotherapy is administered when breast-conserving treatment is undertaken or a large tumor with affected lymph nodes is present. Hormonal treatment does not have a role, as estrogen receptors are always absent from both tumor components. Chemotherapy is nearly always administered on the basis of estrogen receptor and progesterone negativity and the more aggressive potential of the non-adenoid cystic component. The de-differentiation of an indolent type of cancer to a more aggressive one may affect the prognosis.     

Fungal functional ecology: bringing a trait‐based approach to plant‐associated fungi

Fungi play many essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through the soil, water and atmosphere, and are major regulators of macro‐organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function . Trait‐based approaches have been instrumental in strengthening our understanding of plant functional ecology and, as such, provide excellent models for deepening our understanding of fungal functional ecology in ways that complement insights gained from traditional and ‐omics‐based techniques. In this review, we synthesize current knowledge of fungal functional ecology, taxonomy and systematics and introduce a novel database of fungal functional traits (Fun^Fun). Fun^Fun is built to interface with other databases to explore and predict how fungal functional diversity varies by taxonomy, guild, and other evolutionary or ecological grouping variables. To highlight how a quantitative trait‐based approach can provide new insights, we describe multiple targeted examples and end by suggesting next steps in the rapidly growing field of fungal functional ecology.     

Synthesis of homoagmatine and GC–MS analysis of tissue homoagmatine and agmatine: evidence that homoagmatine but not agmatine is a metabolite of pharmacological L-homoarginine in the anesthetized rat

Amino Acids - Low L-homoarginine (hArg) concentrations in human blood and urine are associated with renal and cardiovascular morbidity and mortality, yet the underlying mechanisms and the...