Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry

Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.     

Perchlorate binding to cytochrome c: a magnetic and optical study

1. The effects of perchlorate on cytochrome c have been investigated by 1H and 35Cl NMR, electron paramagnetic resonance and optical spectroscopy. 2. The pK values for the formation and disappearance of the major alkaline conformation were found to be displaced from 9.3 to 8.3 and from 10.4 to 10.9, respectively. The displacement was dependent on the ClO4(-) concentration below 0.1 M. 3. Competition experiments between perchlorate and chloride show that ClO4(-) binds both to the neutral and alkaline forms but with a higher affinity for the latter. The appearance of a new binding site in the alkaline form accounts for the markedly enhanced relaxation rate of 35ClO4(-) in this pH range. Complex formation between cyanide and the alkaline species results in the loss of this binding site, which probably is located close to or within the heme crevice. 4. The neutral form of ferricytochrome c also binds perchlorate strongly as evidence by the unique appearance of a high-spin signal dependent on pH and perchlorate concentration. This signal disappears with the same pK value as the neutral form. The effects of perchlorate on cytochrome c are due to specific binding of this ion.     

Cerebral asymmetry in Old World monkeys.

A photogrammetric computer analysis of 88 endocasts, representing 8 genera of Old World monkeys, reveals cortical asymmetry in lengths of the Sylvian fissure, superior temporal sulcus, lateral edge of the orbit and distance separating rectus and arcuate sulci. Hypothetical expansion of left prefrontal and parietal integration cortices explains these asymmetries.     

Chromoplasts of Tropaeolum majus L.: Structure and development

Summary The fine structure of chromoplasts in epidermal cells of flower petals of Tropaeolum has been investigated by light, polarizing, and electron microscopy at different stages of development. The pale greenish-yellow petals still enclosed in the bud contain barely differentiated chloroplasts with few, irregular grana. The chromoplasts of unfolding petals show differently oriented bundles of tubules with variable diameters (mean: 17 nm). Thylakoid membranes become reduced more and more. The tubular bundles are intermingled with numerous isodiametric bodies of ca. 50 nm diameter; these bodies are better discernible at later stages when the chromoplasts possess a less dense matrix. The chromoplasts of open flowers are in a state of disorganization at a time when the cytoplasm still appears normal. A comparison is made between chromoplast tubules and tubular structures described from other kinds of plastids. The observations are discussed in view of chromoplast typology and with regard to possible processes underlying chromoplast differentiation in flowers.Abbreviations in Figures Chr chromoplast - CT chromoplast tubules - Cy cytoplasm - D dictyosome - IB isodiametric body - M mitochondrion - MT microtubule - oG osmiophilic globule - S S-body - St starch grain - V vacuole All micrographs from glutaraldehyde-OsO₄-fixed material, unless otherwise specified. The bar designates 1 m (multiples or fractions of it indicated).     

Synthetic flavinyl peptides related to the active site of mitochondrial monoamine oxidase. I. Chemical and spectral properties.

Various 8 alpha-sulfur-linked peptides related to the flavinyl peptides isolated from mitochondrial monoamine oxidase were synthesized in high yield and purity. The peptides, protected by an acetyl-blocking group on the amino terminus, were synthesized by conventional liquid-phase techniques and coupled to a tetraacetylriboflavin derivative activated in the 8alpha position. In some cases, the ribityl side chains of the flavinyl peptides were selectively deacetylated. In other cases, the thioether functions were oxidized to form sulfones. These flavinyl peptides were studied by uv-visible absorption and circular dichroic spectroscopies. A close correspondence in spectroscopic and other chemical properties indicated the identity of the synthetic and naturally obtained flavinyl peptides. Differences between the tetraacetylriboflavinyl and riboflavinyl peptides indicate an interaction between the ribityl side chain and thioether function in aqueous media. Evidence was obtained for an intramolecular complex between the tyrosyl and isoalloxazine moieties in aqueous media. Substitution in the 8alpha position was accompanied by an impairment of the protonation of the N1 position of the isoalloxazine ring and a lowering of the redox potential relative to the parent 8-methyflavins.     

Redistribution of the Fc receptor on human blood monocytes and peritoneal macrophages induced by immunoglobulin G-sensitized erythrocytes.

The Fc receptor is a plasma membrane component exhibiting an affinity for the C gamma 3 domain of certain subclasses of immunoglobulin G. Using anti-Rho (D)-sensitized red cells (EA) in a slide rosette technique, we have demonstrated a translational mobility and polar redistribution of this receptor on the human blood monocyte and peritoneal macrophage. These cells, isolated from venous blood and malignant ascites and identified histochemically, showed a time- and temperature-dependent receptor capping defined by binding six or more EA confined to the cell half-perimeter. Caps formed in serum were mainly extreme caps in which bound EA appeared as a morula contiguous with the adherent cell. At 21 degrees C and 37 degrees C in serum 80% live rosettes formed caps; virtually none formed at 4 degrees C and about 25% were seen in PBS at 21 degrees C. Similarly, 10% extreme caps in PBS and 60 and 70% in serum were seen at room temperature and 37 degrees C, respectively, suggesting a serum factor(s) was important in promoting live rosette capping. Capping was reversibly inhibited by sodium azide although inhibition was incomplete even at 0.1 M, a concentration 10-fold higher than that giving complete inhibition of lymphocyte antigen-receptor capping. Azide also induced reversion of capped rosettes to diffuse, noncapped rosettes, and to levels comparable to those seen in inhibition studies. Re-exposure to EA after rosette capping failed to increase either the proportion of cells forming rosettes or the fullness of such rosettes indicating a critical number of receptors had been capped. Live rosettes induced a spherocytosis in bound EA irrespective of capping status; such changes appeared early in PBS where capping was minimal. Dead cells bore EA as normal biconcave discs. Some rosetting EA were ultimately hemolyzed upon prolonged incubation, and erythrophagocytosis was seen in about 15% of capped rosettes at 37 degrees C.     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.     

Receptor for advanced glycation end products is subjected to protein ectodomain shedding by metalloproteinases

The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a gamma-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by gamma-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.     

The characterization of chromosome breaks in Drosophila melanogaster. II. Molecular analysis of gamma-ray-induced deficiencies in the 14F-15A region

In the previous paper of this series, a total of 446 mutations of the para gene were isolated following gamma- or X-irradiation. Of these, 180 were shown to be chromosome deficiencies. In this analysis we examine the molecular distribution of breakpoints in a subset of strains [38] which have an endpoint in the 14F-15A5 region. We find that although the breakpoints are distributed throughout the entire region, there is some regional specificity to the distribution of those endpoints.