Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.     

Suppression of mutations in the core of the Tetrahymena ribozyme by spermidine, ethanol and by substrate stabilization.

We have previously described a collection of mutations in conserved residues of the core of the Tetrahymena self-splicing intron. Most of these single base substitutions have less than 10% of the activity of their parental intron derivative [Couture, S., et al., (1990) J. Mol. Biol., 215, 345-358]. We examined the effect of two agents known to stabilize RNA structure, spermidine and ethanol, on the activity of many of these mutant RNAs. In the presence of either 5 mM spermidine or 20% ethanol most substitution mutations were partially or completely suppressed. These conditions also increased the temperature optima of both wild-type and mutant ribozymes. In addition, we find that mutations are also suppressed by a high concentration of GTP, a substrate in the reaction which is bound specifically by the intron. Thus we observe a general suppression of mutations in an RNA enzyme (ribozyme) by spermidine, ethanol and by substrate stabilization. These results are consistent with the idea that most mutations destabilize the folded structure of the ribozyme and can be suppressed by any of a variety of stabilizing influences.     

A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density.

The growth rates and buoyant densities of a Salmonella typhimurium mutant, TL126 (proB74A+), with enhanced osmotolerance caused by proline overproduction were measured and compared with the growth rates and buoyant densities of an isogenic (wild-type) strain, TL128 (proB+ A+), with normal control of proline production. Growth rates were determined in a rich medium (Luria broth) with added NaCl to produce various osmotic strengths ranging from 300 to 2,000 mosM. At low concentrations of NaCl, there was little variation in doubling times between the two strains. However, as the osmotic strength of the medium approached and exceeded 1,300 mosM, the doubling times of TL126 (osmotolerant) were 1.5 to 2 times faster than those of TL128 (wild type), confirming the osmotolerance of TL126. Buoyant densities were determined by equilibrium sedimentation in a Percoll gradient of osmotic strength equal to that of the growth medium. The osmolarity of the Percoll gradient was adjusted by the addition of NaCl. At low osmolarities (300 to 500 mosM), the buoyant density of TL126 (osmotolerant) was slightly but consistently lower than that of TL128 (wild type). As the osmotic strength was increased, the buoyant density of TL126 (osmotolerant) increased in proportion to the osmotic strength. In contrast, the buoyant density of strain TL128 (wild type) did not increase as much. At high osmolarities (1,600 to 2,000 mosM), the buoyant density of TL126 (osmotolerant) was consistently higher than that of TL128 (wild type). These results suggest that the intracellular accumulation of proline by TL126, the osmotolerant strain, increases both the growth rates and buoyant densities at osmolarities of 1,300 mosM and above.     

Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.     

Molecular markers shared by diverse apomictic Pennisetum species

Two molecular markers, a RAPD (randomly amplified polymorphic DNA) and a RFLP/STS (restriction fragment length polymorphism/sequence-tagged site), previously were found associated with apomictic reproductive behavior in a backcross population produced to transfer apomixis from Pennisetum squamulatum to pearl millet. The occurrence of these molecular markers in a range of 29 accessions of Pennisetum comprising 11 apomictic and 8 sexual species was investigated. Both markers were specific for apomictic species in Pennisetum. The RFLP/STS marker, UGT 197, was found to be associated with all taxa that displayed apomictic reproductive behavior except those in section Brevivalvula. Neither UGT197 nor the cloned RAPD fragment OPC-04₆₀₀ hybridized with any sexually reproducing representatives of the genus. The cloned C04₆₀₀ was associated with 3 of the 11 apomictic species, P. ciliare, P. massaicum, and P. squamulatum. UGT197 was more consistently associated with apomictic reproductive behavior than OPC04₆₀₀ or cloned C04₆₀₀, thus it could be inferred that UGT197 is more closely linked to the gene(s) for apomixis than the cloned C04₆₀₀. The successful use of these probes to survey other Pennisetum species indicates that apomixis is a trait that can be followed across species by using molecular means. This technique of surveying species within a genus will be useful in determining the relative importance of newly isolated markers and may facilitate the identification of the apomixis gene(s).     

Root movements: Towards an understanding through attempts to model the processes involved

Roots have the ability to change the direction of their forward growth. Sometimes these directional changes are rapid, as in mutations, or they are slower, as in tropisms. The gravitational force is always present and roots have an efficient graviperception mechanism which enables them to initiate gravitropic movements. In trying to model and simulate the course of gravitropic root movements with a view to analyse the component processes, the following aspects of the plant's interaction with gravity have been considered: (1) The level of organization (organism, organ, cell) at which the movement process is expressed; (2) whether the gravity stimulation event is dynamic or static (i.e. whether or not physiologically significant displacements take place with respect to the gravity vector); (3) the sub-systems involved in movement and the processes which they regulate; (4) the mathematical characterization of the relevant sub-systems. A further allied topic is the nature of nutational movements and whether they are linked with gravitropic movements in some way. In considering how they can best be modelled, two types of nutational movements are proponed: stochastic nutation and circumnutation. Most, if not all, natural movements developed in response to static gravistimulation can be viewed as gravimorphisms. This applies at the levels of cell, organ and organism. However, when a system at any one of these levels experiences dynamic gravistimulation, because of its inherent homeostatic properties, it is induced to regenerate a state similar to that previously held. Thus, gravitropism is a regenerative gravimorphic process at the level of the organ.     

Regulatory implications of translational frameshifting in cellular gene expression

The genetic code, once thought to be rigid, hag been found to be quite fiexible, permitting several different reading alternatives. One of these is translatlonal frameshifting, a process programmed in the mRNA sequence and which enables a +1 or -1 shift from the reading frame of the initiation codon. So far, the Involvement of translatlonal frameahifting in gene expression has been described mainly in viruses (particularly retroviruses), retrotransposons, and bacterial insertion elements, in this MicroReview., we present a survey of the cellular genes, mostly in Escherichia coil, which have been found to be expressed through a transiational frameshifting process, as well as a discussion of the regulatory implications of this process.     

Molecular and genetic characterisation of German families with paramyotonia congenita and demonstration of founder effect in the Ravensberg families

Eighteen German families with a history of paramyotonia congenita (PC) were characterised by genetic und mutational analysis at the SCN4A locus, which encodes the -subunit of the adult skeletal muscle sodium channel. We concentrated our analysis primarily on these families to test the hypothesis that a predominance of one common mutation occurs in all German PC families and that this mutation arose in a common ancestor originating in the North-West of the country. The present eighteen PC families exhibit two different mutations (R1448C and R1448H) on various SCN4A dinucleotide repeat haplotypes and therefore the majority of the mutations probably occurred independently. However, the R1448H mutation is extremely frequent in the North-West of Germany (Ravensberger Land) on a specific SCN4A microsatellite haplotype, indicating a founder effect within this subpopulation. Our results suggest that the R1448C/R1448H mutations are by far the most common to be associated with the PC phenotype in the German population.This paper is dedicated to Professor Dr. Dr. Peter Emil Becker on the occasion of this 85th birthday     

Direct determination of the enantiomeric purity of (R)- and (S)-2-hydroxy-4-phenylbutyric acid

The determination of enantiomeric purity of (R)- and (S)-2-hydroxy-4-phenylbutyric acid by chiral HPLC is described. Good resolution has been obtained on covalently bonded L-hydroxyproline saturated with Cu(II) ions. The method makes possible the determination of enantiomeric purity in media containing growing cells. © 1994 Wiley-Liss, Inc.     

Origin of the main class of repetitive DNA within selected Pennisetum species

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 by and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 by and 160 by KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 by KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 by KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 by KpnI families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 by KpnI repeat probably evolved from the 160 by KpnI repeat since the missing 18 by segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.Florida Aqricultural Experiment Station series #R-02758