Recombinant Arabidopsis SQD1 converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro

The sulfolipid sulfoquinovosyldiacylglycerol is a component of plant photosynthetic membranes and represents one of the few naturally occurring sulfonic acids with detergent properties. Sulfolipid biosynthesis involves the transfer of sulfoquinovose, a 6-deoxy-6-sulfoglucose, from UDP-sulfoquinovose to diacylglycerol. The formation of the sulfonic acid precursor, UDP-sulfoquinovose, from UDP-glucose and a sulfur donor is proposed to be catalyzed by the bacterial SQDB proteins or the orthologous plant SQD1 proteins. To investigate the underlying enzymatic mechanism and to elucidate the de novo synthesis of sulfonic acids in biological systems, we developed an in vitro assay for the recombinant SQD1 protein from Arabidopsis thaliana. Among different possible sulfur donors tested, sulfite led to the formation of UDP-sulfoquinovose in the presence of UDP-glucose and SQD1. An SQD1 T145A mutant showed greatly reduced activity. The UDP-sulfoquinovose formed in this assay was identified by co-chromatography with standards and served as substrate for the sulfolipid synthase associated with spinach chloroplast membranes. Approximate K(m) values of 150 microm for UDP-glucose and 10 microm for sulfite were established for SQD1. Based on our results, we propose that SQD1 catalyzes the formation of UDP-sulfoquinovose from UDP-glucose and sulfite, derived from the sulfate reduction pathway in the chloroplast.     

The formation of stable fatty acid substrate complexes in prostaglandin H(2) synthase-1

We have developed a protocol to purify apo-ovine (o) prostaglandin endoperoxide H(2) synthase-1 (PGHS-1) to homogeneity from ram seminal vesicles. The resulting apo enzyme can then be reconstituted with Co(3+)-protoporphyrin IX instead of Fe(3+)-protoporphyrin IX to produce a native-like, but functionally inert, enzyme suitable for the production of enzyme:fatty acid substrate complexes for biophysical characterization. Co(3+)-protoporphyrin IX reconstituted oPGHS-1 (Co(3+)-oPGHS-1) displays a Soret band at 426 nm that shifts to 406 nm upon reduction. This behavior is similar to that of cobalt-reconstituted horseradish peroxidase and myoglobin and suggests, along with resonance Raman spectroscopy, that the Co(3+)-protoporphyrin IX group is one in a six-coordinate, cobalt(III) state. However, Co(3+)-oPGHS-1 does not display cyclooxygenase or peroxidase activity, nor does the enzyme produce prostaglandin products when incubated with [1-(14)C]arachidonic acid. The cocrystallization of Co(3+)-oPGHS-1 and the substrate arachidonic acid (AA) has been achieved using sodium citrate as the precipitant in the presence of the nonionic detergent N-octyl-beta-d-glucopyranoside. Crystals are hexagonal, belonging to the space group P6(5)22, with cell dimensions of a = b = 181.69 A and c = 103.74 A, and a monomer in the asymmetric unit. GC-MS analysis of dissolved crystals indicates that unoxidized AA is bound within the crystals.     

Stretch-shortening cycle exercises: an effective training paradigm to enhance power output of human single muscle fibers.

Functional performance of lower limb muscles and contractile properties of chemically skinned single muscle fibers were evaluated before and after 8 wk of maximal effort stretch-shortening cycle (SSC) exercise training. Muscle biopsies were obtained from the vastus lateralis of eight men before and after the training period. Fibers were evaluated regarding their mechanical properties and subsequently classified according to their myosin heavy chain content (SDS-PAGE). After training, maximal leg extensor muscle force and vertical jump performance were improved 12% (P<0.01) and 13% (P<0.001), respectively. Single-fiber cross-sectional area increased 23% in type I (P<0.01), 22% in type IIa (P<0.001), and 30% in type IIa/IIx fibers (P<0.001). Peak force increased 19% in type I (P<0.01), 15% in type IIa (P<0.001), and 16% in type IIa/IIx fibers (P<0.001). When peak force was normalized with cross-sectional area, no changes were found for any fiber type. Maximal shortening velocity was increased 18, 29, and 22% in type I, IIa, and hybrid IIa/IIx fibers, respectively (P<0.001). Peak power was enhanced in all fiber types, and normalized peak power improved 9% in type IIa fibers (P<0.05). Fiber tension on passive stretch increased in IIa/IIx fibers only (P<0.05). In conclusion, short-term SSC exercise training enhanced single-fiber contraction performance via force and contraction velocity in type I, IIa, and IIa/IIx fibers. These results suggest that SSC exercises are an effective training approach to improve fiber force, contraction velocity, and therefore power.     

Sequential detection of antigens in Western blots with differently colored products

A reliable sequential application of peroxidase and alkaline phosphatase-conjugated second antibodies to detection of two different antigens in Western blots is described. Deposits of differently colored products formed allow identification of several antigens on a single blot.     

NHERF links the N-cadherin/catenin complex to the platelet-derived growth factor receptor to modulate the actin cytoskeleton and regulate cell motility

Using phage display, we identified Na+/H+ exchanger regulatory factor (NHERF)-2 as a novel binding partner for the cadherin-associated protein, beta-catenin. We showed that the second of two PSD-95/Dlg/ZO-1 (PDZ) domains of NHERF interacts with a PDZ-binding motif at the very carboxy terminus of beta-catenin. N-cadherin expression has been shown to induce motility in a number of cell types. The first PDZ domain of NHERF is known to bind platelet-derived growth factor-receptor beta (PDGF-Rbeta), and the interaction of PDGF-Rbeta with NHERF leads to enhanced cell spreading and motility. Here we show that beta-catenin and N-cadherin are in a complex with NHERF and PDGF-Rbeta at membrane ruffles in the highly invasive fibrosarcoma cell line HT1080. Using a stable short hairpin RNA system, we showed that HT1080 cells knocked down for either N-cadherin or NHERF had impaired ability to migrate into the wounded area in a scratch assay, similar to cells treated with a PDGF-R kinase inhibitor. Cells expressing a mutant NHERF that is unable to associate with beta-catenin had increased stress fibers, reduced lamellipodia, and impaired cell migration. Using HeLa cells, which express little to no PDGF-R, we introduced PDGF-Rbeta and showed that it coimmunoprecipitates with N-cadherin and that PDGF-dependent cell migration was reduced in these cells when we knocked-down expression of N-cadherin or NHERF. These studies implicate N-cadherin and beta-catenin in cell migration via PDGF-R-mediated signaling through the scaffolding molecule NHERF.     

Aldosterone influences free intracellular calcium in human mononuclear leukocytes in vitro

Mineralocorticoid receptors have been detected in human mononuclear leukocytes (HML) and a physiological effector mechanism was demonstrated subsequently by which aldosterone is able to prevent the loss of intracellular sodium, potassium and cell water during incubation in an aldosterone-free medium. In the present paper, free intracellular calcium, [Ca2+]i, was measured in HML from normal subjects by Quin-2 and Fura-2 fluorescence after incubation for 1 h at 37 degrees C in RPMI-1640 medium. In fresh HML, [Ca2+]i was 54 +/- 15 nM (Fura-2, mean +/- SD, n = 26). After incubation without aldosterone, [Ca2+]i in HML was 118 +/- 27 nM (Quin-2, n = 11) and 50 +/- 13 nM (Fura-2). After incubation with 1.4 (Fura-2) or 2.8 nM (Quin-2) aldosterone, [Ca2+]i was 139 +/- 38 nM (Quin-2, P less than 0.05 compared with value after incubation without aldosterone) and 57 +/- 11 nM (Fura-2, P less than 0.00001). The Kd-value for dose-response curve was 0.4 nM. The effect of aldosterone was antagonized by N-ethyl-isopropylamiloride, but not by canrenoate, canrenone, cycloheximide and actinomycin D. It was absent in a sodium-free buffer. Corticosterone and hydrocortisone were active as agonists. These results show that aldosterone exerts an effect on the [Ca2+]i in HML in vitro which could be involved in hemodynamic responses to mineralocorticoids if also present in cardiovascular tissues.     

Structure and Development of the Olfactory Organ in the Garfish Belone belone (L.) (Teleostei,Atheriniformes)

Theisen, B., Breucker, H., Zeiske, E., Melinkat, R. 1980. Structure and development of the olfactory organ in the garfish Belone belone (L.) (Teleostei, Atheriniformes). (Institute of Comparative Anatomy, University of Copenhagen, Denmark; Anatomisches Institut, Universität Hamburg, and Zoologisches Institut und Zoologisches Museum, Universität Hamburg, Federal Republic of Germany.) — Acta zool. (Stockh.) 61(3): 161–170. The structure and development of the olfactory organ in the garfish Belone belone (L.) were studied by light and electron microscopy (SEM and TEM). The olfactory organ has the shape of an open groove with a protruding papilla. In embryos and early juveniles the groove is smooth and is provided with a continuous sensory epithelium. During ontogenesis the papilla develops and the composition of the epithelium is changed as areas of nonsensory epithelium appear and eventually separate the sensory epithelium into islets. In adults the sensory epithelium consists of supporting, basal, and two types of receptor cells, ciliated and microvillous. In juveniles also ciliated nonsensory cells are present. This difference can be correlated with differing locomotory habits of adults and juveniles. The receptor cilia show a 9 + 0 microtubular pattern while the nonsensory cilia have the general 9 + 2 pattern. Deviating dendritic endings were found and are considered an indication of ongoing cell dynamics.     

Unusual muscle abnormalities associated with thalidomide treatment in a rhesus monkey: a case report.

Unusual musculotendinous abnormalities were observed in the forelimbs of a rhesus monkey treated prenatally with thalidomide (10 mg/kg maternal body weight, gestation days 33, 34, and 35). Although the hindlimbs exhibited malformations typical of thalidomide dysmelia, the forelimbs appeared relatively normal externally. However, dissection revealed totally anomalous insertions of preaxial muscles of both forearms. Extensor muscles inserted on flexor surfaces even though normal sites of insertion were present and unoccupied. Radiographs revealed only minor ossification deficiencies in the carpus and distal radius. These observations suggest that thalidomide, given at the appropriate time, can disrupt condensation and splitting of pre-muscle masses without greatly disturbing condensation of corresponding preskeletal elements. These types of musculoskeletal relationships are unusual findings in congenital malformations of the limbs and have not been widely associated with thalidomide teratogenesis.