Sister chromatid exchange analysis of the 15q11 region in Prader-Willi syndrome patients

Summary Prader-Willi syndrome (PWS) is a sporadic disorder in which about half of cases have a 15q12 deletion. Although a small number of cases have other rearrangements involving 15q12, the rest of the cases appear to have normal chromosomes. Clinical similarities among all these patients regardless of the karyotype strongly suggests a common etiology. To investigate the nature of this common etiology, we analyzed sister chromatid exchange (SCE) at the 15q11-13 region in 10 PWS patients with the chromosome deletion, 12 PWS patients with normal chromosomes, and 11 normal control individuals. While SCE at the q11-13 region was absent on the 15q12 deleted chromosome, the percentage of SCE on chromosome 15 at q11 was statistically higher for PWS with normal chromosomes (10.1%) compared to that for normal controls (1.9%) and the normal homologue (2.2%) in deleted patients (²=7.7982, df=2, P<0.025). The data suggest relative instability of DNA at the 15q11 region in PWS patients.     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Effect of immune system imagery on secretory IgA

This study was an investigation of the effects of physiologically-oriented mental imagery on immune functioning. College students with normal medical histories were randomly selected to one of three groups. Subjects in Group 1 participated in short educational training on the production of secretory immunoglobulin A. They were then tested on salivary IgA, skin temperature, and the Profile of Mood States (POMS) before and after listening to a 17-minute tape of imagery instructions with specially composed background entrainment music designed to enhance imagery. Subjects in Group 2 (placebo controls) listened to the same music but received nor formal training on the immune system. Group 3 acted as a control and subjects were tested before and after 17 minutes of no activity. Treatment groups listened to their tapes at home on a bi-daily basis for six weeks All groups were again tested at Weeks 3 and 6. Secretory IgA was analyzed using standard radial immunodiffusion techniques. Repeated measures analyses of variance with planned orthogonal contrasts were used to evaluate the data. Significant overall increases (p<0.05) were found between pre- and posttests for all three trials. Groups 1 and 2 combined (treatment groups) yielded significantly greater increases in sIgA over Group 3 (control) for all three trials. Group 1 (imagery) was significantly higher than Group 2 (music) in antibody production for Trials 2 and 3. Symptomatology, recorded by subjects at Weeks 3 and 6, was significantly lower for three symptoms (rapid heartbeat, breathing difficulty, and jaw clenching), favoring both treatment groups over the control group.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Identification of phytochrome B amino acid residues mutated in three new phyB mutants of Arabidopsis thaliana

The growth and development of plants is regulated by light viathe action of photoreceptors which are responsive to the red/far-red,blue and UV regions of the spectrum. Phytochrome B (the apoproteinof which is encoded by the PHYB gene) is one of the red/far-redabsorbing photoreceptors active in this process. In this paper,the isolation and characterization of three new EMS-inducedmutations of Arabidopsis which confer phytochrome B deficiencyare described. Complementation analysis showed that these mutations(phyB-101, phyB-102 and phyB-104) were allelic with PHYB. DNAsequence analysis showed that all three mutants contain nucleotidesubstitutions in the PHYB-101 gene sequence. phyB-101 carriesa nucleotide substitution within the second exon of the PHYBgene. This G-to-A substitution is a missense mutation that convertsa glutamate residue at position 812 of the phytochrome B apoproteinto a lysine residue. phyB-102, another missense mutant, carriesa C-to-T substitution which converts a serine residue at position349 of the phytochrome B apoprotein to a phenylalanine residue.phyB-104 carries a premature stop codon as a result of a G-to-Amutation 1190 bp down-stream of the ATG start codon of the PHYBsequence. The missense mutations in phyB-101 and phyB-102 causesignificant alterations in the predicted second ary structureof their respective mutant polypeptides, and identify aminoacid residues playing crucial roles in phytochrome B function,assembly or stability. Key words: Arabidopsis thaliana, phytochromet, phyB mutants, missense mutations     

Responses of nitrate assimilation and N translocation in tomato (Lycopersicon esculentum Mill) to reduced ambient air humidity

The impact of low humidity in ambient air on water relations,nitrate uptake, and translocation of recently absorbed nitrogen,was investigated in 5-week-old tomato (Lycopersicon esculentumMill cv. Ailsa Craig) plants grown hydroponically in a completenutrient solution. Plants were subjected to dry air (relativehumidity 2–4% for 6 h. The transpiration rate increasedseveral-fold and the shoot water content decreased by almost20%, whereas root water content was unaffected. No effect onin vitro nitrate reductase (NR) activity was detected when usingan EDTA-contraining assay buffer. Replacement of EDTA with Mg²⁺revealed a significant decline in shoot NR activity, which suggestsphosphorylation of the enzyme during the stress treatment. Plantswere grown in a split-root system, in which one root half wasfed ¹⁵N-nitrate during the treatment, in order to determinenitrate uptake and translocation of recently absorbed nitrogenin the plants. Uptake of nitrate was substantially inhibited,but the proportion of absorbed ¹⁵N that was translocated tothe shoots was only slightly affected. In untreated plants,71% of the ¹⁵N recovered in roots had been retranslocated fromthe shoots, whereas in plants subjected to stress the deliveryof ¹⁵N from shoots to roots appeared to be completely inhibited.The data show that lowered humidity in air has significant effectson both uptake of nitrate as well as translocation of nitrogenwithin the plants. Some of these effects appear to be commonwith those observed in plants subjected to reduced water potentialsin the root environment and point to the possibility of theshoot water relations being highly influential on nitrogen uptakeand translocation. Key words: Air humidity, nitrate assimilation, nitrate reductase activity, nitrogen translocation, tomato, water stress     

Modulation of ζ-Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ζ-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ζ-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

Extracellular Matrix Penetration by Epithelial Cells Is Influenced by Quantitative Changes in Basement Membrane Components and Growth Factors

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substratein vitroto form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin–nidogen complex to the substrate or by addition of TGFβ2 to the culture medium, but not TGFβ1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.     

Alarm in a wasp-wasp nesting association: Do members signal cross-specifically?

Summary We studied interspecific alarm communication between two species of social wasps in a nesting association in northwestern Costa Rica by testing the alarm responses ofPolybia occidentalis andMischocyttarus immarginatus to venom of both species. This is the first investigation of alarm pheromone in the genusMischocyttarus. M. immarginatus did not respond with alarm behavior to the venom of either species.P. occidentalis, which responds to its own venom with alarm behavior (Jeanne, 1981), also responded with alarm behavior to the venom ofM. immarginatus, but with much less intensity. Since heterospecific venoms did not release species-typical levels of alarm behavior, we conclude that interspecific alarm signalling plays no role in the nesting association.