Local-scale Arctic tundra heterogeneity affects regional-scale carbon dynamics.

In northern Alaska nearly 65% of the terrestrial surface is composed of polygonal ground, where geomorphic tundra landforms disproportionately influence carbon and nutrient cycling over fine spatial scales. Process-based biogeochemical models used for local to Pan-Arctic projections of ecological responses to climate change typically operate at coarse-scales (1km2-0.5°) at which fine-scale (<1km2) tundra heterogeneity is often aggregated to the dominant land cover unit. Here, we evaluate the importance of tundra heterogeneity for representing soil carbon dynamics at fine to coarse spatial scales. We leveraged the legacy of data collected near Utqiagvik, Alaska between 1973 and 2016 for model initiation, parameterization, and validation. Simulation uncertainty increased with a reduced representation of tundra heterogeneity and coarsening of spatial scale. Hierarchical cluster analysis of an ensemble of 21st-century simulations reveals that a minimum of two tundra landforms (dry and wet) and a maximum of 4km2 spatial scale is necessary for minimizing uncertainties (<10%) in regional to Pan-Arctic modeling applications.

Comparison of cryopreservation methods on T-cell responses to islet and control antigens from type 1 diabetic patients and controls.

BACKGROUND: Type 1 diabetes (T1D) is a cell-mediated autoimmune disease characterized by destruction of the pancreatic islet cells. The use of cryopreserved cells is preferable to the use of freshly isolated cells to monitor clinical trials to decrease assay and laboratory variability. METHODS: The T-Cell Workshop Committee of the Immunology of Diabetes Society compared two widely accepted T-cell freezing protocols (warm and cold) to freshly isolated peripheral blood mononuclear cells from patients with T1D and controls in terms of recovery, viability, cell subset composition, and performance in functional assays currently in use in T1D-related research. Nine laboratories participated in the study with four different functional assays included. RESULTS: The cold freezing method yielded higher recovery and viability compared with the warm freezing method. Irrespective of freezing protocol, B cells and CD8+ T cells were enriched, monocyte fraction decreased, and islet antigen-reactive responses were lower in frozen versus fresh cells. However, these results need to take in to account that the overall response to islet autoantigens was low in some assays. CONCLUSIONS: In the current study, none of the tested T-cell functional assays performed well using frozen samples. More research is required to identify a freezing method and a T-cell functional assay that will produce responses in patients with T1D comparable to responses using fresh peripheral blood mononuclear cells.

The hemoregulatory peptide pEEDCK may inhibit stem cell proliferation via hydropathic binding to antisense sequence motifs in interleukin-11 and other growth factors.

In undisturbed bone marrow, most hemopoietic stem cells are nonproliferating despite the presence of multiple growth factors. Endogenous inhibitory factors are responsible for maintenance of this quiescence. Previously we sequenced and synthesized the inhibitory pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK), which originally derives from granulocytes, and investigated the role of this peptide in stem cell quiescence. To provide some mechanistic insight, in the present work we studied the structural relationship of this peptide to specific growth-factor-derived sequence motifs. In the murine system in vivo as well as in long-term bone marrow, antiserum to pEEDCK produced a significant stimulation of formation of colony-forming units-granulocyte/macrophage. Binding of peptides to proteins often takes place at hydropathically complementary sites. Therefore, we searched for peptides corresponding to the complementary sequence to pEEDCK. We identified antisense sequences in the genes of various cytokines and cytokine receptors including interleukin-11. The corresponding peptide Val-Leu-Leu-Thre-Arg (VLLTR) and several other peptides hydropathically complementary to pEEDCK were synthesized. We found that pEEDCK binds specifically to these peptides as well as to complete interleukin-11. Dissociation constants were in the 10 microM range. The peptide hydropathically corresponding to pEEDCK (VLLTR) was found to stimulate colony-forming units-granulocyte/macrophage formation. Our data suggest that pEEDCK could exert a coordinating function in the hemopoietic cytokine network by binding to multiple regulatory proteins and modulating their activity.

Mutation of the acetylcholine receptor epsilon-subunit promoter in congenital myasthenic syndrome.

Congenital myasthenic syndrome comprises a heterogeneous group of inherited disorders of neuromuscular transmission. Acetylcholine receptor (AChR) deficiency is the most common form of congenital myasthenic syndrome and in most cases results from mutations within the coding region of the AChR epsilon subunit. However, studies in mice have established that synapse-specific expression of AChR is dependent on a sequence contained within the AChR-subunit promoter regions, termed an N-box. We describe a consanguineous family in which 2 of 7 siblings had clinical and electromyographic features consistent with AChR deficiency. Muscle biopsy demonstrated low AChR numbers, establishing the disorder as postsynaptic. Single-strand conformational polymorphism analysis identified an abnormal conformer in the AChR epsilon-subunit gene promoter of the patients. DNA sequence and restriction endonuclease analysis shows that the disorder cosegregates with recessive inheritance of a single point mutation, a transition (C-->T) in the N-box of the epsilon-subunit promoter. Analysis of an intercostal biopsy from 1 of the patients showed a dramatic reduction in epsilon-subunit mRNA levels compared with disease and normal controls. This is the first evidence in humans that an N-box mutation can lead to disruption of epsilon-subunit transcription, resulting in the loss of adult AChR synthesis and the clinical phenotype of AChR-deficiency congenital myasthenic syndrome.

Correction of Class II, Division 2 malocclusions through the use of the Bionator appliance. Report of two cases.

The correction of two Class II, Division 2 malocclusions during the mixed dentition phase with the use of a Bionator appliance is presented. The suggestion that correction of Class II, Division 2 malocclusions may be achieved in the absence of fixed appliances is supported in these case reports.

Localization of nucleocapsid associated polypeptides in measles virus-infected cells by immunogold labelling after resin embedding.

The nucleo-, phospho- and matrix protein of measles virus were localized at high resolution within infected cells by use of post-embedding immunogold labelling techniques. In general, labelling with monospecific antibodies as well as with a polyvalent rabbit anti-measles hyperimmune antiserum revealed measles virus polypeptides to be distributed non-randomly within infected cells with the label largely confined to specific sites, namely inclusions of nucleocapsids and assembled virus structures at the plasma membrane. Immunogold double labelling indicated that the phosphoprotein strictly co-localized with the nucleoprotein in cytoplasmic inclusions of nucleocapsids and in budding virions, whereas intranuclear inclusions of nucleocapsids were devoid of phosphoprotein labelling. Antibodies to the matrix protein clearly labelled assembled virus structures at the plasma membrane but exhibited no significant cytoplasmic or intranuclear reaction. The data indicate that the composition of nucleocapsids varies with the cellular compartment with which they are associated, supporting the view of a rapid assembly of paramyxovirus nucleocapsid polypeptides, and emphasize the proposed selective role of the matrix protein in virus assembly and budding at the plasma membrane.

Vascular growth factors and the development of macrovascular disease in diabetes mellitus.

The pathogenesis of macrovascular disease in diabetes mellitus is still incompletely understood. Within the various pathomechanisms abnormal growth of vascular cells is well established as an intrinsic part of the angiopathic process. In this regard, there are different groups of vascular growth factors that are of potential relevance for the development of macrovascular disease in diabetes : hormones, locally released growth factors of platelet and of arterial wall cell origin. The following hormones whose blood levels could increase under various conditions in diabetes have to be considered : growth hormone, insulin-like growth factor I and II and insulin. Human platelets contain at least eight growth peptides or proteins that all stimulate in vitro growth of arterial wall cells : platelet-derived-, epidermal-, fibroblast-, diabetic serum-, endothelial- and transforming growth factor, vascular endothelial cell proliferation factor and platelet-derived endothelial cell mitogen. In serum and plasma from type II diabetics only the diabetic serum growth factor has been shown to be increased. Platelets from type I and II diabetic patients contain increased growth stimulating activity. This increased growth activity returned to normal levels in both types of diabetes after strict metabolic control. Arterial endothelial and smooth muscle cells, fibroblasts and monocyte/macrophages of different species release at least in culture a variety of growth factors that could participate in an autocrine or paracrine manner in the growth regulation of the arterial wall. Diabetes may affect the release of these factors, but direct evidence to which degree this would contribute to the development of macrovascular disease is lacking.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased growth stimulation of human vascular cells by serum from patients with primary hyper-LDL-cholesterolemia.

Premature atherosclerosis in hypercholesterolemic patients may be due, in part, to increased growth of vascular cells. Therefore, the growth stimulating effect of serum and serum fractions from patients with primary hyper-LDL-cholesterolemia (LDL-cholesterol: 7.5 +/- 1.7 mmol/l) and from healthy subjects on human arterial smooth muscle cells and fibroblasts has been investigated over 5-7 days in culture. Human hypercholesterolemic sera increased the growth of both cell types up to a mean of 133% compared with normal sera (100%) (P less than 0.001). Removal of the dialyzable serum fraction (m.w. less than 3,500 daltons) reduced the growth effect of the hypercholesterolemic sera by 32% (P less than 0.001) and of the normal sera by 11% (P less than 0.01). Readdition of the hypercholesterolemic serum dialysate to its dialyzed serum restored completely the original growth effect. There was no significant difference in growth stimulation between the dialyzed hypercholesterolemic and normal sera excluding a major additional growth effect by LDL-cholesterol. The low molecular weight growth factor(s) of hypercholesterolemic serum (m.w. less than 3,500 daltons) showed a linear dependence of growth stimulation over a 20-fold concentration range. Increased amounts of this factor(s) might easily penetrate the arterial wall, thus contributing to atherogenesis.

Sera from type 2 (non-insulin-dependent) diabetic and healthy subjects contain different amounts of a very low molecular weight growth peptide for vascular cells.

Diabetic angiopathy may be due, in part, to increased growth in vascular cells. We have investigated serum growth factors in Type 2 (non-insulin-dependent) diabetic and healthy subjects and their effect on cultured human arterial smooth muscle cells and fibroblasts. Removal of the dialyzable serum fraction (mol. wt. less than 12,000) reduced the growth effect of the diabetic sera by 37% (2p less than 0.005) and of the non-diabetic sera by only 8% (2p less than 0.01). In contrast, there was no difference in growth stimulation between the dialyzed diabetic or non-diabetic sera. Complete recovery of the dialyzable serum growth fraction was also obtained at a mol. wt. below 3,500. Ten times the concentration of the low molecular weight growth factor (mol. wt. less than 3,500) from diabetic sera stimulated growth of fibroblasts or arterial smooth muscle cells by a mean of 243% or 174% and from non-diabetic sera by a mean of 146% or 137%, respectively (2p less than 0.01). The growth stimulating potency of this serum fraction (mol. wt. less than 3,500) contained in 10% diabetic sera, was two to ten times higher than that of human growth hormone or insulin, added in amounts equivalent to 10% or physiological serum concentrations. This diabetic serum growth factor was further characterized by: (1) linear dependence of growth stimulation over a concentration range of twenty times and by (2) reduction of the growth stimulating activity to control levels by pretreatment: (a) at 95 degrees C for 30 min, or (b) with two different proteases: Serva pronase E (Streptomyceus griseus) or Calbiochem protease (Subtilisin calsberg).(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular growth factors and the development of macrovascular disease in diabetes mellitus.

There are two different classes of humoral growth factors for arterial smooth muscle and endothelial cells that age of potential relevance for the development of macrovascular disease inn diabetes mellitus: hormones (growth hormone, insulin like growth factor I and II, insulin) and locally released growth factors of platelet origin. The following hormones have to be considered: Increased growth hormone plasma levels might contribute to macrovascular disease, but its actual relevance remains to be determined. Insulin like growth factor I and II are present in vivo and stimulate growth of vascular cells in vitro but their relevance for macrovascular disease in diabetes is unproven. To insulin, see Dr. Stout's paper. Human platelets contain at least six growth peptides or proteins that all stimulate in vitro growth of arterial wall cells: platelet derived growth factor, epidermal growth factor, platelet derived endothelial cell mitogen, endothelial growth factor, diabetic serum growth factor (DSGF), transforming growth factor-beta. As their plasma concentrations have not been shown to be increased in diabetes increased local availability at sites of stimulated platelet aggregation has been postulated. Therefore, their relevance for macrovascular disease i diabetics is based mainly on circumstantial evidence. The concentration of DSGF of platelet origin depends on the metabolic control: it increases in vivo in poorly controlled diabetics and is normalized after 2-3 weeks of good metabolic control in the same diabetic patient. The growth potency of DSGF from poorly controlled diabetics is greater than that of physiological amounts of insulin or growth hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloroperoxidase compound I: Electron paramagnetic resonance and Mössbauer studies.

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. Mössbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and Mössbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)

Mössbauer and electron paramagnetic resonance studies of horseradish peroxidase and its catalytic intermediates.

We report Mössbauer and EPR measurements on horseradish peroxidase in the native state and the reaction intermediates with peroxide and chlorite. A detailed analysis of the electronic state of the heme iron is given, and comparisons are drawn with related systems. The native enzyme is high-spin ferric and thus has three Kramers doublets. The unusual magnetic properties of the ground doublet and the large energy of the second, (E2-E1)/k approximately equal to 41 K, and third doublet, (E3-E1)/k greater than or equal to 170 K, can be modeled with a quartet admixture of approximately 11% to the spin sextet. All evidence suggests a ferryl, OFeIV, state of the heme iron in compounds I and II and related complexes. The small isomer shift, delta Fe approximately equal to 0.06 mm/s, the (positive) quadrupole splitting, delta EQ approximately equal to 1.4 mm/s, the spin S = 1, and the large positive zero field splitting, D/k approximately equal to 35 K, are all characteristic of the ferryl state. In the green compound I the iron weakly couples to a porphyrin radical with spin S' = 1/2. A phenomenological model with a weak exchange interaction S . J . S', magnitude of less than or equal to 0.1 D, reproduces all Mössbauer and EPR data of compound I, but the structural origin of the exchange and its apparent distribution require further study. Reaction of horseradish peroxidase with chlorite leads to compound X with delta Fe = 0.07 mm/s and delta EQ = 1.53 mm/s, values that are closest to those of compound II. The diamagnetism of compound III and its Mössbauer parameters delta Fe = 0.23 mm/s and delta EQ = -2.31 mm/s at 4.2 K clearly identify it as an oxyheme adduct.

Chemical nature of the porphyrin pi cation radical in horseradish peroxidase compound I.

The electron paramagnetic resonance (EPR) and Mössbauer properties of native horseradish peroxidase have been compared with those of a synthetic derivative of the enzyme in which a mesohemin residue replaces the natural iron protoporphyrin IX heme prosthetic group. The oxyferryl pi cation radical intermediate, compound I, has been formed from both the native and synthetic enzyme, and the magnetic properties of both intermediates have been examined. The optical absorption characteristics of compound I prepared from mesoheme-substituted horseradish peroxidase are different from those of the compound I prepared from native enzyme [DiNello, R. K., & Dolphin, D. (1981) J. Biol. Chem. 256, 6903-6912]. By analogy to model-compound studies, it has been suggested that these optical absorption differences are due to the formation of an A2u and an A1u pi cation radical species, respectively. However, the EPR and Mössbauer properties of the native and synthetic enzyme and of their oxidized intermediates are quite similar, if not identical, and the data favor an A2u radical for both compounds I.

Zero-field splitting of Fe3+ in horseradish peroxidase and of Fe4+ in horseradish peroxidase compound I from electron spin relaxation data.

From the temperature dependence of the Orbach relaxation rate of the paramagnetic center in horseradish peroxidase (HRP), we deduce an excited-state energy of 40.9 +/- 1.1 K. Similar studies on the broad EPR signal of HRP compound I indicate a much weaker Orbach relaxation process involving an excited state at 36.8 +/- 2.5 K. The strength of the Orbach process in HRP-I is weaker than one would normally estimate by 2-4 orders of magnitude. This fact lends support to the model of HRP-I involving a spin 1/2 free radical coupled to a spin 1 Fe4+ heme iron via a weak exchange interaction. Such a system should exhibit an Orbach relaxation process involving delta E, the excited state of the Fe4+ ion, but reduced in strength by (Jyy/delta E)2, where Jyy is related to the strength of the exchange interaction between the two spin systems.

The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I.

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.

Kinetic analysis of compound I formation and the catalatic activity of chloroperoxidase.

When the only substrate added to a solution of chloroperoxidase is a hydroperoxide, the reactions are: ferric enzyme + ROOH leads to compound I + ROH and compound I + ROOH leads to ferric enzyme + O2 + ROH. When H2O2 is used as substrate, the rate constants for the formation and catalatic decomposition of compound I are 2.4 X 10(6) M--1.S--1 and 3.4 X 10(5) M--1.S--1 at pH 4.7 and it is predicted that a maximum of 87% of the enzyme converts to compound I in the steady state of the catalatic reaction. With methyl hydroperoxide, formation of compound I has a rate constant of 4.7 X 10(5) M--1.S--1 and its decomposition 2.9 X 10(4) M--1.S--1. When peracetic acid is used, compound I is formed with a rate constant of 3.8 X 10(6) M--1.S--1 and a 100% yield of compound I is obtained.