Horseradish peroxidase-catalyzed oxidation of chlorophyll a with hydrogen peroxide: Characterization of the products and mechanism of the reaction

Horseradish peroxidase was verified to catalyze, without any phenol, the hydrogen peroxide oxidation of chlorophyll a (Chl a), solubilized with Triton X-100. The 13²(S) and 13²(R) diastereomers of 13²-hydroxyChl a were characterized as major oxidation products (ca. 60%) by TLC on sucrose, UV-vis, ¹H, and ¹³C NMR spectra, as well as fast-atom bombardment MS. A minor amount of the 15²-methyl, 17³-phytyl ester of Mg-unstable chlorin was identified on the basis of its UV-vis spectrum and reactivity with diazomethane, which converted it to the 13¹,15²-dimethyl, 17³-phytyl ester of Mg-purpurin 7. The side products (ca. 10%) were suggested to include the 17³-phytyl ester of Mg-purpurin 18, which is known to form easily from the Mg-unstable chlorin. The side products also included two red components with UV-vis spectral features resembling those of pure Chl a enolate anion. Hence, the two red components were assigned to the enolate anions of Chl a and pheophytin a or, alternatively, two different complexes of the Chl a enolate ion with Triton X-100. All the above products characterized by us are included in our published free-radical allomerization mechanism of Chl a, i.e. oxidation by ground-state dioxygen. The HRP clearly accelerated the allomerization process, but it did not produce bilins, that is, open-chain tetrapyrroles, the formation of which would require oxygenolysis of the chlorin macrocycle. In this regard, our results are in discrepancy with the claim by several researchers that ‘bilirubin-like compounds’ are formed in the HRP-catalyzed oxidation of Chl a. Inspection of the likely reactions that occurred on the distal side of the heme in the active centre of HRP provided a reasonable explanation for the observed catalytic effect of the HRP on the allomerization of Chl. In the active centre of HRP, the imidazole nitrogen of His-42 was considered to play a crucial role in the C-13² deprotonation of Chl a, which resulted in the Chl a enolate ion resonance hybrid. The Chl enolate was then oxidized to the Chl 13²-radical while the HRP Compound I was reduced to Compound II. The same reactive Chl derivatives, i.e. the Chl enolate anion and the Chl 13²-radical, which are produced twice in the HRP reaction cycle, happen to be the crucial intermediates in the initial stages of the Chl allomerization mechanism.     

VCP Associated Inclusion Body Myopathy and Paget Disease of Bone Knock-In Mouse Model Exhibits Tissue Pathology Typical of Human Disease

Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget''s disease of bone and frontotemporal dementia (IBMPFD). We have generated a knock-in mouse model with the common R155H mutation. Mice demonstrate progressive muscle weakness starting approximately at the age of 6 months. Histology of mutant muscle showed progressive vacuolization of myofibrils and centrally located nuclei, and immunostaining shows progressive cytoplasmic accumulation of TDP-43 and ubiquitin-positive inclusion bodies in quadriceps myofibrils and brain. Increased LC3-II staining of muscle sections representing increased number of autophagosomes suggested impaired autophagy. Increased apoptosis was demonstrated by elevated caspase-3 activity and increased TUNEL-positive nuclei. X-ray microtomography (uCT) images show radiolucency of distal femurs and proximal tibiae in knock-in mice and uCT morphometrics shows decreased trabecular pattern and increased cortical wall thickness. Bone histology and bone marrow derived macrophage cultures in these mice revealed increased osteoclastogenesis observed by TRAP staining suggestive of Paget bone disease. The VCP^R155H/+ knock-in mice replicate the muscle, bone and brain pathology of inclusion body myopathy, thus representing a useful model for preclinical studies.     

Regulation of action potential duration under acute heat stress by I(K,ATP) and I(K1) in fish cardiac myocytes

The mechanism underlying temperature-dependent shortening of action potential (AP) duration was examined in the fish (Carassius carassius L.) heart ventricle. Acute temperature change from +5 to +18 degrees C (heat stress) shortened AP duration from 2.8 +/- 0.3 to 1.3 +/- 0.1 s in intact ventricles. In 56% (18 of 32) of enzymatically isolated myocytes, heat stress also induced reversible opening of ATP-sensitive K+ channels and increased their single-channel conductance from 37 +/- 12 pS at +8 degrees C to 51 +/- 13 pS at +18 degrees C (Q10 = 1.38) (P < 0.01; n = 12). The ATP-sensitive K+ channels of the crucian carp ventricle were characterized by very low affinity to ATP both at +8 degrees C [concentration of Tris-ATP that produces half-maximal inhibition of the channel (K1/2)= 1.35 mM] and +18 degrees C (K1/2 = 1.85 mM). Although acute heat stress induced ATP-sensitive K+ current (IK,ATP) in patch-clamped myocytes, similar heat stress did not cause any glibenclamide (10 microM)-sensitive changes in AP duration in multicellular ventricular preparations. Examination of APs and K+ currents from the same myocytes by alternate recording under current-clamp and voltage-clamp modes revealed that changes in AP duration were closely correlated with temperature-specific changes in the voltage-dependent rectification of the background inward rectifier K+ current IK1. In approximately 15% of myocytes (4 out of 27), IK,ATP-dependent shortening of AP followed the IK1-induced AP shortening. Thus heat stress-induced shortening of AP duration in crucian carp ventricle is primarily dependent on IK1. IK,ATP is induced only in response to prolonged temperature elevation or perhaps in the presence of additional stressors.     

Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces

In this study we showed that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. These covalent bonds had no major effects on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci, which have native --> denatured transition midpoints (T(m)) of 98.6 and 94.7 degrees C, respectively, in the absence of biotin, will find use in applications where extreme stability or minimal leakage of subunits is required. Furthermore, we showed that the intramonomeric disulfide bridges found in the wild-type avidin affect its stability. The mutant Avd-nc, in which this bridge was removed, had a lower T(m) in the absence of biotin than the wild-type avidin but showed comparable stability in the presence of biotin.     

An improved method of computing the wear factor for total hip prostheses involving the variation of relative motion and contact pressure with location on the bearing surface

A new method of computing the wear factor for total hip prostheses is presented. In the conventional method, only the resultant contact force and the track drawn by the point of its application are considered so that the product of the instantaneous force and sliding increment is integrated over one motion cycle. In the present, improved, method the contact pressure distribution is discretized by a large number of smaller normal forces, and the contribution of each is summed. This is important because the relative motion and contact pressure vary strongly with location, and because the transverse pressure component is substantial. Hence, the present surface integral represents the large contact surface better than the conventional line integral. A prerequisite for the surface integral was the method of computing the relative motion correctly anywhere on the contact surface, developed and published earlier by the present authors. For the pressure discretization, the contact surface was divided into nearly equal-sized surface elements. The contact pressure was modelled with ellipsoidal, paraboloidal and sinusoidal distributions. Two load cases were studied, double-peak and static. When an ellipsoidal contact pressure distribution extending over a hemisphere was discretized by 1000 element forces, the computed wear factor for double-peak load in a biaxial hip wear simulator was 30% lower than in the conventional resultant force case. The present method can be later developed further to involve the temporal variation of size and location of the contact surface.     

A new locus for coeliac disease mapped to chromosome 15 in a population isolate

Coeliac disease is a common multifactorial disease with a strong genetic component, which is not entirely explained by the HLA association. Four previous whole-genome screens have produced somewhat inconsistent results suggesting genetic heterogeneity. We attempted to overcome this problem by performing a genome-wide scan in a Finnish sub-population, expected to be more homogeneous than the general population of Finland. The families in our study originate from the northeastern part of Finland, the Koilliskaira region, which has been relatively isolated since its founding in the 16th century. Genealogical studies have confirmed that the families share a common ancestor in the 16th century. Nine families with altogether 23 patients were genotyped for 399 microsatellite markers and the data were analysed with parametric linkage analysis using two dominant and one recessive model. A region on chromosome 15q11-q13 was implicated with a LOD score of 3.14 using a highly penetrant dominant model. Addition of more markers and one more sib-pair increased the LOD score to 3.74. This result gives preliminary evidence for existence of a susceptibility factor in this chromosomal region.     

Endogenous plasma membrane t-SNARE syntaxin 4 is present in rab11 positive endosomal membranes and associates with cortical actin cytoskeleton

Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 4 is a t-SNARE that, according to previous overexpression studies, is predominantly localized at the plasma membrane. In the present study endogenous syntaxin 4 was found in intracellular vesicular structures in addition to regions of the plasma membrane. In these vesicular structures syntaxin 4 colocalized with rab11, a marker of recycling endosomes. Furthermore, syntaxin 4 colocalized with actin at the dynamic regions of the plasma membrane. Treatment with N-ethylmaleimide, the membrane transport inhibitor, caused an increased accumulation of syntaxin 4/rab11 positive vesicles in actin filament-like structures. Finally, purified recombinant syntaxin 4 but not syntaxin 2 or 3 cosedimented with actin filaments in vitro, suggesting direct interaction between these two proteins. Taken together, these data suggest that syntaxin 4 regulates secretion at the actin-rich areas of the plasma membrane and may be recycled through rab11 positive intracellular membranes.     

Refuge sites of voles under owl predation risk: priority of dominant individuals?

In an aviary experiment, we studied whether body size or habitatfamiliarity of field voles (Microtus agrestis) affected predationrisk by Tengmalm's owls (Aegolius funereus). In the field, wecompared the body size of field voles snap-trapped in good (covered)and poor (open) habitats in 1992 and 1994 to determine whetherthere were habitat-related differences in the body size of voles.In the aviary, large individuals occupied the good habitat significantlymore than small individuals both in the control (owl not present)and experimental treatments (owl present). Furthermore, habitat-familiarvoles inhabited the good habitat more than habitat-unfamiliarvoles did when an owl was present Our field data were consistentwith our aviary data: larger field voles were more frequentlyfound in good habitats than in poor habitats. In the aviary,Tengmalm's owl predation risk was higher for small and habitat-unfamiliarvoles. This suggests that large field voles may have priorityto sheltered habitats. Furthermore, habitat familiarity mayplay a central role in avoiding risky habitats.