Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction

The kinetics of the light-induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin-transducin coupling.     

Enzyme assay of L-myo-inositol 1-phosphate synthetase based on high-performance liquid chromatography of benzoylated inositol

A procedure for the determination of inositol by reversed-phase HPLC is described which is based on a precolumn benzoylation and detection at 230 nm. This procedure was used to assay the activity of L-myo-inositol 1-phosphate synthetase (EC 5.5.1.4) after treatment of the enzymatic product by a phosphatase.     

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.     

Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in endothelial cells stimulated with bradykinin

Bovine pulmonary artery endothelial cells (BPAEC) were prelabeled with [3H]choline or [3H]myristic acid to selectively label endogenous phosphatidylcholine. BPAEC were stimulated with ATP and bradykinin (BK), and phospholipase D (PLD) activation was detected as a 4-fold increase in [3H]choline in cells prelabeled with [3H]choline or as a 2- to 3-fold increase in [3H]phosphatidylethanol in cells prelabeled with [3H]myristic acid and stimulated in the presence of ethanol. Pretreatment of BPAEC with 0.1 microM phorbol 12-myristate 13-acetate (PMA) for 22 hr completely inhibited agonist-induced PLD activation, whereas prostacyclin synthesis and [3H]phosphoinositide ([3H]PIns) hydrolysis were enhanced in pretreated cells. Long-term PMA treatment thus dissociates agonist-induced PLD activation from [3H]PIns hydrolysis, and agonist-induced prostacyclin synthesis is not dependent upon PLD activation.     

Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport

N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme's microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.     

DoesAgapetus fuscipes cultivate algae in its case?

The presence of algae within the cases ofAgapetus fuscipes was investigated. Cases recognised as dirty or clean with the naked eye had more and less algal growth, respectively. Larvae in the former survived significantly longer when starved in the laboratory. It is suggested that the presence of algae within the cases would be of ecological advantage during periods of flood.     

Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.     

Correlation of spinule dynamics and plasticity of the horizontal cell spectral response in cyprinid fish retina: quantitative analysis

Summary A negative feedback interaction between luminosity type horizonatal cells (HCs) and green-sensitive cones generates the long-wavelength-sensitive depolarizing response in biphasic chromaticity type HCs. This interaction is suppressed in the dark and is potentiated by light adaptation of the retina. HCs are morphologically plastic; during light adaptation, their dendritic terminals within cone pedicles extend, giving rise to spinules. This paper examines whether there is a quantitative correlation between the time course of light-dependent formation of the spinules and enhancement of the feedback interaction. The strength of the feedback interaction in isolated retinac of the roach was determined as the neutral wavelength at which reversal of spectral response polarity occurred in biphasic HCs. A good correlation was found between the neutral wavelength and the spinule/ribbon ratios of retinae. Biphasic HCs were intracellularly stained with horseradish peroxidase and the correlative ultrastructure of the contacted pedicles was examined. Neutral wavelength was found to be correlated with the spinule number, weighted according to the number of synaptic contacts mediating feed-forward transmission. The latter was estimated from the total number of labelled C_b/H2 HC processes (central and lateral) at synaptic triads. A model in which spinules mediate the negative feedback interaction of HCs in the retina of cyprinid fish is presented.