Ethylene biosynthesis in isolated vacuoles of Vicia faba L. — requirement for membrane integrity

The ability of vacuoles prepared from V. faba leaves to convert 1-aminocyclopropane-1-carboxylic acid to C₂H₄ was destroyed when vacuoles were lysed by passage through a hypodermic needle, freezing and thawing, osmotic shock, treatment with ethanol or with a detergent. Ethylene synthesis in the vacuolar fraction was also inhibited by the uncouplers carbonyl cyanide m-chlorophenyl hydrazone and dinitrophenol and by the ionophores valinomycin, nigericin, and A23187. Ethylene formation increased with increasing pH of the incubation medium over the pH range of 5.0–7.5. These observations support the hypothesis that C₂H₄ biosynthesis in vacuolar preparations is dependent on membrane integrity, possibly because of the requirement for a transmembrane ion gradient.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenyl hydrazone     

Delipidated retroviruses as potential autologous therapeutic vaccines--a pilot experiment

This pilot experiment in a simian immunodeficiency virus (SIV) chronic infection model aimed at extending our previous findings that vaccination with delipidated SIV resulted in more potent and diversified antiviral responses (1). Macaques chronically infected with SIVmac239 treated with antiretroviral therapy (ART) were vaccinated with autologous delipidated virus via consecutive lymph node targeted immunizations-1, 1 and 10 mug of virus spaced monthly. Results showed all animals had lasting viral load reduction approaching 1 log compared to set-point, and disease delay. Delipidation may enhance processing/ presentation of viral antigen eliciting potent antiviral control even at such late infection stage.     

The Synthesis of Collagen and Glycosaminoglycans by Dedifferentiated Chondroblasts in Culture

Marked changes occur in the morphology of chick chondroblasts grown for 5 days in F-10 medium containing either 5-bromo-2'-deoxyuridine or embryo extract. The cells lose the characteristic polygonal morphology and assume a flattened 'fibroblastic' appearance. To determine whether the morphological changes reflect a biochemical transformation toward frank fibroblasts, changes in collagen and glycosaminoglycan synthesis were examined in these 'dedifferentiated' cells. Growth in either medium did not significantly affect the total amount of collagen synthesized. However, the subunit composition of the collagen chains was different. Freshly isolated cartilage trunks or control chondroblast cultures synthesized only α1 subunits (suggesting exclusive synthesis of α1(ll)₃-type collagen), whereas dedifferentiated cultures synthesized both α1 and, in addition, some α2 subunits (suggesting synthesis of fibroblasttype α1(l)₂α2-type collagen). Incorporation of labelled glucosamine in F-10 medium showed that the major glycosaminoglycan synthesized by either cartilage trunks or chondroblast monolayers is chondroitin sulphate; little, if any, hyaluronic acid could be detected. With growth in embryo extract (EE) glucosamine was incorporated equally into chondroitin sulphate and hyaluronic acid, whereas in BUdR, chondroitin sulphate synthesis was completely inhibited. Distinct biochemical differences were therefore found for both collagen and glycosaminoglycan synthesis during growth in either BUdR or EE. Such changes were not identical but both demonstrate changes in synthetic programme tending to approach that of frank fibroblasts.     

In vitro attachment of skeletal muscle fibers to a collagen gel duplicates the structure of the myotendinous junction

The myotendinous junction (MTJ) and its associated cells and connective tissue are important structures involved in transmission of contractile force from skeletal muscle to tendon. A model culture system was developed to investigate the formation of the MTJ and its attachment to collagen fibers. Skeletal muscle cells were cultured in a well modeled from two layers of a native gel of type I collagen. Muscle cells cultured in this manner formed attachments to the collagen gel and developed into highly contractile multinucleated muscle fibers with the development of extensive terminal invaginations of the sarcolemma. In addition, the subsarcolemma at the ends of muscle fibers showed areas of increased electron density which corresponded well with the termini of myofibrils. The results indicate that the development of sarcolemmal invaginations at the end of a muscle fiber probably occurs intrinsically during muscle development in vivo. The direct association of collagen fibers with the basal lamina at the end of muscle fibers was only occasionally observed in culture, suggesting that other fibrils or proteins may also be involved in the attachment of collagen fibers to the basal lamina of muscle fibers at the MTJ.     

Characterization of the collagen chains synthesized by cultured smooth muscle cells derived from rhesus monkey thoracic aorta.

Five different collagen chains and one smaller collagenous fragment have been isolated from the collagens found in the combined cell layer and medium of rhesus monkey aortic smooth muscle cell cultures. The collagen chains which can be identified are alpha1 (III), alpha1(I), alpha2, A and B. The smaller collagenous peptide exhibits an apparent molecular weight of 45 000 and has been designated CP45 (Mayne, R., et al. (1977), Arch. Biochem. Biophys. 181, 462). Smooth muscle cells continue to synthesize the collagens from which these components are derived for at least eight passages in culture. At each passage the alpha1 (III) chain consistently represents about one-half of the total collagen which is recovered after initial fractionation by agarose gel chromatography. The results show that smooth muscle cells derived from rhesus monkey thoracic aorta are phenotypically stable for many generations in vitro.     

Serum Iron Levels and the Risk of Parkinson Disease: A Mendelian Randomization Study

Background

Although levels of iron are known to be increased in the brains of patients with Parkinson disease (PD), epidemiological evidence on a possible effect of iron blood levels on PD risk is inconclusive, with effects reported in opposite directions. Epidemiological studies suffer from problems of confounding and reverse causation, and mendelian randomization (MR) represents an alternative approach to provide unconfounded estimates of the effects of biomarkers on disease. We performed a MR study where genes known to modify iron levels were used as instruments to estimate the effect of iron on PD risk, based on estimates of the genetic effects on both iron and PD obtained from the largest sample meta-analyzed to date.

Methods and Findings

We used as instrumental variables three genetic variants influencing iron levels, HFE rs1800562, HFE rs1799945, and TMPRSS6 rs855791. Estimates of their effect on serum iron were based on a recent genome-wide meta-analysis of 21,567 individuals, while estimates of their effect on PD risk were obtained through meta-analysis of genome-wide and candidate gene studies with 20,809 PD cases and 88,892 controls. Separate MR estimates of the effect of iron on PD were obtained for each variant and pooled by meta-analysis. We investigated heterogeneity across the three estimates as an indication of possible pleiotropy and found no evidence of it. The combined MR estimate showed a statistically significant protective effect of iron, with a relative risk reduction for PD of 3% (95% CI 1%–6%; p = 0.001) per 10 µg/dl increase in serum iron.

Conclusions

Our study suggests that increased iron levels are causally associated with a decreased risk of developing PD. Further studies are needed to understand the pathophysiological mechanism of action of serum iron on PD risk before recommendations can be made. Please see later in the article for the Editors'' Summary     

The foldon substructure of staphylococcal nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔG_HX) and the kinetic unfolding and refolding rates (k_op and k_cl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 × 10^− 6 s^− 1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.     

Relationship Between Changes in EMG and Respiratory Sinus Arrhythmia in a Study of Relaxation Therapy for Asthma

This paper reports the relationships among changes in cardiovagal activity, surface EMG, and measures of pulmonary function in a study of relaxation therapy for asthma. Changes in FEV ₁ /FVC were negatively correlated with those in cardiac interbeat interval, consistent with the hypothesis that relaxation-induced changes in airway function are mediated autonomically, with increased vagal tone and/or decreased sympathetic arousal producing bronchoconstriction. Contrary to Kotses's theory of a vagal-trigeminal reflex as mediator for relaxation-induced improvement in asthma, decreases in pulmonary function occurred during relaxation sessions, accompanied by increases in cardiovagal activity, and within-session changes in frontal EMG in the first session of training were positively associated with changes in a measure of pulmonary function (FEV1/FVC). However, consistent with this hypothesis, first-session frontalis EMG changes were positively associated with changes in respiratory sinus arrhythmia, and last-session changes in cardiac interbeat interval were positively associated with changes in FEV1/FVC. The results suggest that the immediate effects of generalized relaxation instruction can be associated with a parasympathetic rebound, which, in turn, may induce countertherapeutic changes in asthma. However, the effects of specific facial muscle relaxation remain uncelar.