Psychosocial Stress Increases Salivary Alpha-Amylase Activity Independently from Plasma Noradrenaline Levels

Salivary alpha-amylase activity (sAA) and plasma noradrenaline (NA) concentrations are often considered to be surrogate markers of sympathetic activation in response to stress. However, despite accumulating evidence for a close association between sAA and noradrenaline and other indicators of sympathetic activity, reliability and generality of this relation remains unclear. We employed the Trier Social Stress Test (TSST) in order to directly compare the responses in sAA and NA to psychological stress in healthy volunteers (n = 23). The TSST significantly increased sAA and NA plasma levels with no significant differences in females and males. However, when subjects were divided according to their NA responses into low versus high responders, both groups did not significantly differ in their sAA before, during or after stress exposure. These data suggest that in response to acute psychological stress both plasma NA levels and sAA reflect sympathetic activity, however seemed to increase independently from each other.     

Validation of the Fibromyalgia Survey Questionnaire within a cross-sectional survey

The Fibromyalgia Survey Questionnaire (FSQ) assesses the key symptoms of fibromyalgia syndrome. The FSQ can be administrated in survey research and settings where the use of interviews to evaluate the number of pain sites and extent of somatic symptom intensity and tender point examination would be difficult. We validated the FSQ in a cross-sectional survey with FMS patients. In a cross-sectional survey, participants with physician diagnosis of FMS were recruited by FMS-self help organisations and nine clinical institutions of different levels of care. Participants answered the FSQ (composed by the Widespread Pain Index [WPI] and the Somatic Severity Score [SSS]) assessing the Fibromyalgia Survey Diagnostic Criteria (FSDC) and the Patient Health Questionnaire PHQ 4. American College of Rheumatology 1990 classification criteria were assessed in a subgroup of participants. 1,651 persons diagnosed with FMS were included into analysis. The acceptance of the FSQ-items ranged between 78.9 to 98.1% completed items. The internal consistency of the items of the SSS ranged between 0.75-0.82. 85.5% of the study participants met the FSDC. The concordance rate of the FSDC and ACR 1990 criteria was 72.7% in a subsample of 128 patients. The Pearson correlation of the SSS with the PHQ 4 depression score was 0.52 (p<0.0001) and with the PHQ anxiety score was 0.51 (p<0.0001) (convergent validity). 64/202 (31.7%) of the participants not meeting the FSDC criteria and 152/1283 (11.8%) of the participants meeting the FSDC criteria reported an improvement (slightly too very much better) in their health status since FMS-diagnosis (Chi(2)?=?55, p<0.0001) (discriminant validity). The study demonstrated the feasibility of the FSQ in a cross-sectional survey with FMS-patients. The reliability, convergent and discriminant validity of the FSQ were good. Further validation studies of the FSQ in clinical and general population settings are necessary.     

Crystal structure of the Habc domain of neuronal syntaxin from the squid <Emphasis Type="Italic">Loligo pealei</Emphasis> reveals conformational plasticity at its C-terminus

Background

Intracellular membrane fusion processes are mediated by the spatial and temporal control of SNARE complex assembly that results in the formation of a four-helical bundle, composed of one vesicle SNARE and three target membrane SNARE polypeptide chains. Syntaxins are essential t-SNAREs and are characterized by an N-terminal Habc domain, a flexible linker region, a coiled-coil or SNARE motif and a membrane anchor. The N-terminal Habc domain fulfills important regulatory functions while the coiled-coil motif, present in all SNAREs, is sufficient for SNARE complex formation, which is thought to drive membrane fusion.

Results

Here we report the crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei, s-syntaxin. Squid Habc crystallizes as a dimer and the monomer structure consists of a three-helical bundle. One molecule is strikingly similar to mammalian syntaxin 1A while the second one shows a structural deviation from the common fold in that the C-terminal part of helix C unwinds and adopts an extended conformation.

Conclusion

Conservation of surface residues indicates that the cytosolic part of s-syntaxin can adopt an auto-inhibitory closed conformation that may bind squid neuronal Sec1, s-Sec1, in the same manner as observed in structure of the rat nSec1/syntaxin 1A complex. Furthermore, despite the overall structural similarity, the observed changes at the C-terminus of one molecule indicate structural plasticity in neuronal syntaxin. Implications of the structural conservation and the changes are discussed with respect to potential Habc domain binding partners such as Munc13, which facilitates the transition from the closed to the open conformation.

     

Epidermal fatty acid-binding protein is increased in rat lungs following in vivo treatment with keratinocyte growth factor

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.     

Beiträge zur Physiologie der Pigmentbildung in belichteten etiolierten Blättern

Ohne Zusammenfassung     

Natural and artificial routes to pluripotency

Pluripotent cells of the blastocyst inner cell mass (ICM) and their in vitro derivatives, embryonic stem (ES) cells, contain genomes in an epigenetic state that are poised for subsequent differentiation. Their chromatin is hyperdynamic in nature and relatively uncondensed. In addition, a large number of genes are expressed at low levels in both ICM and ES cells. Also, the chromatin of naturally pluripotent cells contains specialized histone modification patterns such as bivalent domains, which mark genes destined for later developmentally-regulated expression states. Female pluripotent cells contain X chromosomes that have yet to undergo the process of X chromosome inactivation. Collectively, these features of very early embyronic chromatin are required for the successful specification and production of differentiated cell lineages. Artificial reprogramming methods such as somatic nuclear transfer (SCNT), ES cell fusion-mediated reprogramming (FMR), and induced pluripotency (iPS) yield pluripotent cells that recapitulate many features of naturally pluripotent cells, including many of their epigenetic features. However, the route to pluripotent epigenomic states in artificial pluripotent cells differs drastically from that of their natural counterparts. Here, we compare and contrast the differing routes to pluripotency under natural and artificial conditions. In addition, we discuss the intrinsically metastable nature of the pluripotent epigenome and consider epigenetic aspects of reprogramming that may lead to incomplete or inaccurate reprogrammed states. Artificial methods of reprogramming hold immense promise for the development of autologous cell graft sources and for the development of cell culture models for human genetic disorders. However, the utility of artificially reprogrammed cells is highly dependent on the fidelity of the reprogramming process and it is therefore critically important to assess the epigenetic similarities between embryonic and induced pluripotent stem cells.     

A complex containing at least one zinc dependent HeLa nuclear protein binds to the intronic (gaa)n block of the frataxin gene

We analyzed HeLa nuclear proteins binding to the (gaa)_n harbouring intron 1 of nine frataxin alleles and characterized the structures of the repeats. Fragments with blocks longer than (gaa)₉ form spontaneously different intramolecular H-y topoisomeres in linear state. The observed triplexes depend on the length of the repeat. Interruption of the perfectly repeated (gaa)_n block entails two structural regions. At least two HeLa nuclear proteins bind to the (gaa)_n fragments resulting in a distinct major retarded complex as revealed by EMSA. One of these proteins is zinc dependent. Importantly, the fragment harbouring (gan)₁₂₁ binds additional proteins. Protein binding appears to be locus specific, and the binding affinity was found to be not random. The affinities of the different target fragments varied by a factor of four. Binding affinities of the fragments were not obviously correlated to differences in the composition of the repeats. DNase I footprinting revealed only weakly protected binding regions, but multiple HS sites in the repeat regions of the fragments. These findings and the fact, that DNA conformers observed in EMSA and electron microscopical experiments bind proteins, lead to the assumption that the proteins recognize, both, B-DNA and triple helical structures, but with different affinity. Possible functions of the proteins are discussed in the context of transformation of triple helical structures into B-DNA and the pathogenesis of FRDA.     

Mapping of mglB, the structural gene of the galactose-binding protein of Escherichia coli

Summary The tetracycline resistance transposon Tn10 was inserted into the E. coli chromosome near mglB550, a structural gene for the galactose-binding protein. P1 transductions established the position of these Tn10 insertions (zee-700, 701, 702::Tn10) close to the genes ptsF, fpk, cdd, mglB550, his, and gatA with 85%–95%, 85%, 36%, 20%–40%, 12%–15%, and 0.5% contransduction frequency. Three factor crosses revealed the relative sequence of the genes as: mglB550, zee-700::Tn10, ptsF, fpk, cdd, his. gatA was found to be 1.3% cotransducible with mglB550. Two Tn10 insertions near gatA were isolated and characterized. One, zef-704::Tn10, was 3% cotransducible with fpk, 8% with mglB550, and 42% with gatA. The other, zef-703::Tn10, was 98% cotransducible with gatA but not with mglB550 or fpk. Neither of these two Tn10 insertions was cotransducible with cdd. Four factor crosses revealed the sequence gatA, zef-704::Tn10, mglB550, fpk.Neither zee-700::Tn10 nor zef-703::Tn10 showed any (0/300) contransduction with either glpT or gyrA. The clockwise order of genes is then: his, cdd, fpk, ptsF, zee-700::Tn10, mglB550, zef-704::Tn10, gatA. With a fix-point for his at 44 min, fpk would be placed at 45 min and mglB550 at 45.5 min. During the course of this work we noticed that the cotransduction frequency between Tn10 insertions and nearby markers tended to increase when new P1 lysates were prepared from freshly reisolated strains. This may indicate loss of nonessential genes adjacent to Tn10 insertions. Using insertion zee-702::Tn10, we isolated deletions extending into an mgl gene other than mglB. Crosses between such a deletion mutant and an mglB550 mutant were done. The analysis of the periplasmic proteins of these as well as other transductants or recombinants involving the mglB550 or the mglB551 gene revealed the existence of strains synthesizing both the wild-type as well as the corresponding mutant protein. Strains containing both proteins exhibit either wild-type or mutant phenotype. These strains appeared unstable. Upon reisolation from purified stock cultures kept in glycerol at-20°C, colonies could be isolated that carried only mutant or wild-type protein.     

Mucosal layers and related nerve fibres in non-chagasic and chagasic human colon—a quantitative immunohistochemical study

Chagasic megacolon is accompanied by extensive myenteric and, simultaneously, moderate submucosal neuron loss. Here, we examined changes of the innervation pattern of the lamina propria (LP) and muscularis mucosae (MM). Two alternating sets of cryosections were taken from seven non-chagasic colonic and seven chagasic megacolonic specimens (the latter included both the dilated megacolonic and the non-dilated transitional oral and anal zones) and were immunohistochemically triple-stained for smooth-muscle actin (SMA), synaptophysin (SYN) and glial acid protein S100 and, alternatively, for SMA, vasoactive intestinal peptide (VIP) and somatostatin (SOM). Subsequent image analysis and statistical evaluation of nervous tissue profile areas revealed that, in LP, the most extreme differences (i.e. increase in thickness or decrease in nerve, glia and muscle tissue profile area, respectively) compared with control values occurred in the dilated megacolonic zone itself. In contrast, the most extreme differences in the MM were in the anal-to-megacolonic zone (except the profile area of muscle tissue, which was lowest in the megacolonic zone). This parallels our previous results in the external muscle coat. A partial and selective survival of VIP-immunoreactive in contrast to SOM-immunoreactive nerve fibres was observed in both mucosal layers investigated. Thus, VIPergic nerve elements might be crucial for the maintenance of the mucosal barrier. The differential changes of neural tissue parameters in LP and MM might reflect a multifactorial rather than a pure neurogenic development of megacolon in chronic Chagas’ disease.