New evidence for gel-liquid crystalline phase coexistence in the ripple phase of phosphatidylcholines

Experimental evidence supporting the hypothesis of gel-liquid crystalline phase coexistence in the stable ripple phase of diacylphosphatidylcholines has been obtained from time-resolved X-ray small- (SAXS) and wide-angle diffraction (WAXS) in the millisecond to second time domain. The pretransition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) exhibits a thin lamellar liquid crystalline intermediate phase (designated L_α*) if driven far away from equilibrium by an infrared temperature jump (T-jump) technique. The findings can be described by a two-step model. (1) Instantaneously with the T-jump, an anomalously thin lamellar liquid crystalline intermediate phase (d = 5.6–5.8 nm) forms, coexisting with the original gel-phase L_β′. Within the first seconds, the lamellar repeat distance of the intermediate increases to a value of about 6.7 nm. A closer examination of these kinetics reveals two relaxation components: a fast process, proceeding within tenths of a second, and a slow process, on the time scale of a few seconds. (2) Finally, both the liquid crystalline and the gel-phase relax into the stable ripple phase P_β′. The total process time of the transition is nearly independent of the addition of NaCl, but varies strongly with the chain length of the lecithin species. Received: 24 November 1999 / Revised version: 25 February 2000 / Accepted: 25 February 2000     

Zur Oekologie und Brutbiologie des Rohrschwirls(Locustella luscinioides)

Zusammenfassung In den Jahren 1952 bis 1957 wurde die Entwicklung des Rohrschwirl-Bestandes an den Netteseen (Kreis Kempen-Krefeld) verfolgt. Die Siedlungsdichte erreichte 1956 mit 51 singenden ihren Höchststand.Die Ankunft erfolgte im ersten Aprildrittel, der Nestbau begann im letzten Aprildrittel. Alle aufgefundenen Nester standen inCladium-Horsten. Vollgelege wurden erst nach dem 10. Mai gefunden. Brutdauer 12–13 Tage, Nestlingsdauer 14–16 Tage. Beide Eltern waren am Nestbau und bei der Aufzucht der Jungen beteiligt. Der sichere Nachweis einer Zweitbrut gelang nicht; aber einige Beobachtungen deuten darauf hin, daß Zweitbruten vorkommen.     

Tracking the evolution of porphobilinogen synthase metal dependence in vitro

Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+). Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+). The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination. Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges.     

Paradoxical attenuation of leukocyte rolling in response to ischemia- reperfusion and extracorporeal blood circulation in inflamed tissue

In contrast to acute preparations such as the exteriorized mesentery or the cremaster muscle, chronically instrumented chamber models allow one to study the microcirculation under "physiological" conditions, i.e., in the absence of trauma-induced leukocyte rolling along the venular endothelium. To underscore the importance of studying the naive microcirculation, we implanted titanium dorsal skinfold chambers in hamsters and used intravital fluorescence microscopy to study venular leukocyte rolling in response to ischemia-reperfusion injury or extracorporeal blood circulation. The experiments were performed in chambers that fulfilled all well-established criteria for a physiological microcirculation as well as in chambers that showed various extents of leukocyte rolling due to trauma, hemorrhage, or inflammation. In ideal chambers with a physiological microcirculation (<30 rolling leukocytes/mm vessel circumference in 30 s), ischemia-reperfusion injury and extracorporeal blood circulation significantly stimulated leukocyte rolling along the venular endothelium and, subsequently, firm leukocyte adhesion. In contrast, both stimuli failed to elicit leukocyte rolling in borderline chambers (30-100 leukocytes/mm), and in blatantly inflamed chambers with yet higher numbers of rolling leukocytes at baseline (>100 leukocytes/mm), we observed a paradoxical reduction of leukocyte rolling after ischemia-reperfusion injury or extracorporeal blood circulation. A similar effect was observed when we superfused leukotriene B4 (LTB4) onto the chamber tissue. The initial increase in leukocyte rolling in response to an LTB4 challenge was reversed by a second superfusion 90 min later. These observations underscore 1) the benefit of studying leukocyte-endothelial cell interaction in chronically instrumented chamber models and 2) the necessity to strictly adhere to well-established criteria of a physiological microcirculation.     

Histochemical and stereological analysis of the levator ani (pubocaudal) muscle in nulliparous and multiparous beagles

Fiber-type composition and several stereological parameters of the levator ani (pubocaudal) muscle were evaluated in five nulliparous and five multiparous beagles using myosin ATPase-histochemistry and systematically selected muscle cross-sections. With respect to the narrow canine pelvic cavity, this study was also undertaken to determine whether vaginal birth of at least seven litters causes similar neuromuscular changes in the canine levator ani (pubocaudal) muscle analoguous to those seen in the pelvic floor muscles of women after vaginal delivery. The canine pubocaudal muscle is comprised of approximately equal amounts of slow twitch type I and fast twitch type II (IIA, IIS) fibers. The muscles of both the nulliparous and multiparous beagles did not display any signs indicative of denervation or myopathology. The multiparous dogs exhibited significantly increased mean absolute muscle (1720 mm(3)) and total fiber-type I volumes (850 mm(3)) as well as relevantly increased mean diameter of type I fibers (72.0 microm) when compared with the nulliparous group. The canine levator ani (pubocaudal) muscle is not pathologically affected by vaginal deliveries and seems to adapt to numerous successive pregnancies and births through fiber-type I hypertrophy.     

Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans

5-Aminolevulinate synthase (ALAS) is the first and rate-limiting enzyme of heme biosynthesis in humans, animals, other non-plant eukaryotes, and alpha-proteobacteria. It catalyzes the synthesis of 5-aminolevulinic acid, the first common precursor of all tetrapyrroles, from glycine and succinyl-coenzyme A (sCoA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. X-linked sideroblastic anemias (XLSAs), a group of severe disorders in humans characterized by inadequate formation of heme in erythroblast mitochondria, are caused by mutations in the gene for erythroid eALAS, one of two human genes for ALAS. We present the first crystal structure of homodimeric ALAS from Rhodobacter capsulatus (ALAS(Rc)) binding its cofactor PLP. We, furthermore, present structures of ALAS(Rc) in complex with the substrates glycine or sCoA. The sequence identity of ALAS from R. capsulatus and human eALAS is 49%. XLSA-causing mutations may thus be mapped, revealing the molecular basis of XLSA in humans. Mutations are found to obstruct substrate binding, disrupt the dimer interface, or hamper the correct folding. The structure of ALAS completes the structural analysis of enzymes in heme biosynthesis.     

Loss of histochemical identity in mast cells lacking carboxypeptidase A

Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa-/- mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa-/- peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa-/- peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa-/- mice. The Mc-cpa-/- mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.     

Caspase-14 but not caspase-3 is processed during the development of fetal mouse epidermis

The activation of caspases is a central step in apoptosis and may also be critical for terminal differentiation of epidermal keratinocytes (KC). In particular, caspase-3 has been implicated in the differentiation of embryonic KC as well as in programmed cell death of KC, and caspase-14 has been suggested to function in the formation or homeostasis of the stratum corneum (SC). To test the putative roles of these proteases, we determined their expression level and activation status during development of fetal mouse epidermis. The level of procaspase-3 did not change significantly during epidermal development, and enzyme activation was undetectable at any timepoint investigated. Despite the lack of active caspase-3, the newly formed stratum granulosum and the regressing periderm contained cells positive in the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, indicating that nuclear DNA was degraded without activation of caspase-3, thereby arguing against a proteolytic function of caspase-3 in embryonic KC differentiation. By contrast, caspase-14 increased in abundance from embryonic day 14.5 (E14.5) onwards and consistently localized to the suprabasal layers of fetal epidermis. The caspase-14 pro-enzyme was processed into its catalytic subunits, a step required for enzyme activity, on day E17.5, coinciding with SC formation. Thus, processing of procaspase-14 is not confined to air-exposed mature skin but also occurs during epidermal development in utero. In summary, this study demonstrates that caspase-14, but not caspase-3 activation coincides temporally and spatially with embryonic KC differentiation, suggesting a role for caspase-14 in terminally differentiated KC.     

Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound

AIMS: To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. METHOD AND RESULTS: According to plate count results, E. coli (D-value 8.3 min) was far more sensitive than L. rhamnosus (D-value 18.1 min) in their response to the ultrasound intensity applied (20 kHz, 17.6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. CONCLUSIONS: The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.     

MEPD: a resource for medaka gene expression patterns

The Medaka Expression Pattern Database (MEPD) is a database for gene expression patterns determined by in situ hybridization in the small freshwater fish medaka (Oryzias latipes). Data have been collected from various research groups and MEPD is developing into a central expression pattern depository within the medaka community. Gene expression patterns are described by images and terms of a detailed medaka anatomy ontology of over 4000 terms, which we have developed for this purpose and submitted to Open Biological Ontologies. Sequences have been annotated via BLAST match results and using Gene Ontology terms. These new features will facilitate data analyses using bioinformatics approaches and allow cross-species comparisons of gene expression patterns. Presently, MEPD has 19,757 entries, for 1024 of them the expression pattern has been determined.