Modular organization of the multipartite central pattern generator for turtle rostral scratch: knee-related interneurons during deletions.

Central pattern generators (CPGs) are neuronal networks in the spinal cord that generate rhythmic patterns of motor activity in the absence of movement-related sensory feedback. For many vertebrate rhythmic behaviors, CPGs generate normal patterns of motor neuron activities as well as variations of the normal patterns, termed deletions, in which bursts in one or more motor nerves are absent from one or more cycles of the rhythm. Prior work with hip-extensor deletions during turtle rostral scratch supports hypotheses of hip-extensor interneurons in a hip-extensor module and of hip-flexor interneurons in a hip-flexor module.

Variations in motor patterns during fictive rostral scratching in the turtle: knee-related deletions.

Agonist motor neurons usually alternate between activity and quiescence during normal rhythmic behavior; antagonist motor neurons are usually active during agonist motor neuron quiescence. During an antagonist deletion, a naturally occurring motor-pattern variation, there is no antagonist activity and no quiescence between successive bursts of agonist activity. Motor neuron recordings of normal fictive rostral scratching in the turtle displayed rhythmic alternation between activity and quiescence for hip flexors, knee flexors, and knee extensors.

Timing of knee-related spinal neurons during fictive rostral scratching in the turtle.

Knee-flexor motor activity rhythmically alternated with knee-extensor motor activity during fictive rostral scratching in the spinal turtle. A critical transition from knee-flexor motor activity to knee-extensor motor activity occurred during hip-flexor motor activity. A key feature of this transition was that the end-phases of knee-flexor motor activity were positively correlated with the start-phases of knee-extensor motor activity.

Modular organization of turtle spinal interneurons during normal and deletion fictive rostral scratching.

During normal rostral scratching in the spinal turtle, there is rhythmic alternation between hip-flexor and hip-extensor motor activity. During rostral scratching with hip-extensor deletions, there are successive bursts of hip-flexor motor activity and no activity in hip-extensor motor neurons. We characterized the ON- and OFF-phases of 72 descending propriospinal interneurons with distinct activity bursts during normal rostral scratching.

Inducible expression bovine papillomavirus shuttle vectors containing ferritin translational regulatory elements.

The combination of transcriptional and translational control elements in an inducible expression vector suitable for use in stably transformed cell lines was explored. To this end, ferritin translational control elements have been inserted downstream from a mouse metallothionein (mMT-I) transcriptional promoter (PmMT-I), and upstream from various reporter protein-encoding open reading frames (ORFs), all carried on a bovine papillomavirus shuttle vector. Protocols which stimulate transcription (with zinc) and translation (with iron) were developed to optimize the induction of reporter protein synthesis.

Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation.

Induction of ferritin synthesis in cultured cells by heme or iron is accompanied by degradation of the ferritin repressor protein (FRP). Intermediates in the degradative pathway apparently include FRP covalently linked in larger aggregates. The effect of iron on FRP degradation is enhanced by porphyrin precursors but is decreased by inhibitors of porphyrin synthesis, which implies that heme is an active agent.

Specificity of the induction of ferritin synthesis by hemin.

We have previously reported that hemin derepresses ferritin mRNA translation in vitro. As noted earlier, pre-incubation of a 90 kDa ferritin repressor protein (FRP) with hemin prevented subsequent repression of ferritin synthesis in a wheat germ extract. The significance of this observation has been investigated further.

Derepression of ferritin messenger RNA translation by hemin in vitro.

Incubation of a 90-kilodalton ferritin repressor protein (FRP), either free or complexed with an L-ferritin transcript, with hemin or Co3+-protoporphyrin IX prevented subsequent repression of ferritin synthesis in a wheat germ extract. Neither FeCl3 in combinations with H2O2, nor Fe3+ or Fe2+ chelated with EDTA, nor Zn2+-protoporphyrin IX, nor protoporphyrin IX caused significant inactivation of FRP. FRP that had been inactivated by hemin remained chemically intact, as revealed by SDS-polyacrylamide gel electrophoresis.

Requirements for the translational repression of ferritin transcripts in wheat germ extracts by a 90-kDa protein from rabbit liver.

A specific repressor of ferritin mRNA translation originally detected in rabbit reticulocyte lysates has now been purified to homogeneity from rabbit liver, as described in a companion paper (Walden, W. E., Patino, M.

Translational repression in eukaryotes: partial purification and characterization of a repressor of ferritin mRNA translation.

Mouse and rabbit ferritin mRNAs translate very poorly in rabbit reticulocyte lysates relative to most other mRNAs. This translational deficiency is not seen in wheat germ lysates, suggesting the presence of an inhibitor in reticulocyte lysate that is specific for ferritin mRNA. A specific repressor of ferritin mRNA translation has been partially purified from rabbit reticulocytes by differential ultracentrifugation, ammonium sulfate fractionation, and chromatography on phosphocellulose, DEAE-cellulose, and Sephacryl S-300.

Gonadotropin alpha subunit. Differential processing of free and combined forms in human trophoblast and transfected mouse cells.

The gonadotropins luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin are composed of two noncovalently linked subunits, alpha and beta. The alpha subunit, identical in all three hormones, is produced in excess over the unique beta subunits by pituitary and placenta, and is secreted as uncombined, or free subunit. Free alpha subunit from both tissues has a larger molecular weight than the dimer form.

Procedures for enhancing the utility of the metallothionein promoter for the regulated expression of downstream open reading frames.

Procedures which enhance the inducibility of the mouse metallothionein I (mMT-I) transcriptional promoter in mouse C127 cells transformed by bovine papilloma virus have been investigated. These include: (i) induction with Zn2+ at low serum concentration, and (ii) use of a 'superinduction' protocol (presence of 1 microgram/ml of cycloheximide during induction with Zn2+, followed by 2 micrograms/ml of actinomycin D). Use of procedure (i) alone gave a 15- to 20-fold induction of expression of a downstream open reading frame (ORF), which is comparable to the maximum inducibility achieved with mMT-I in other systems.

A map of the hCG beta-LH beta gene cluster.

The beta-subunit of human chorionic gonadotropin (hCG beta) is reportedly encoded by as many as seven non-allelic genes or pseudogenes. Previous studies have identified a cluster of three hCG beta gene copies and the single-copy lutropin-beta subunit (LH beta) gene, but overlap of this cluster with two additional pairs of hCG beta genes has not been demonstrated, despite the isolation of 18 genomic clones. To define the number and organization of non-allelic hCG beta gene copies, genomic Southern blot analyses were performed using hCG beta cDNA and gene-flanking unique-sequence probes.

Shutoff of host translation by encephalomyocarditis virus infection does not involve cleavage of the eucaryotic initiation factor 4F polypeptide that accompanies poliovirus infection.

Studies were conducted to determine whether encephalomyocarditis virus infection causes proteolytic cleavage of any of the polypeptides which comprise eucaryotic initiation factor 4F. Since no such alterations in the components of the initiation factor were detected, these observations confirmed that the mechanisms whereby encephalomyocarditis virus and poliovirus shut off host translation are different.

Unusual requirements for optimum translation of polio viral RNA in vitro.

The translation of poliovirion RNA (polio RNA) in an in vitro fractionated system was much less efficient than that of encephalomyocarditis virion RNA (EMC RNA). In contrast, when polio and EMC RNAs were added to postmitochondrial cell lysates (S10), they were translated with equal efficiency. However, this equality was observed only when high concentrations of S10 were employed; at lower concentrations, polio RNA translation was reduced relative to that of EMC RNA.

Role of mRNA competition in regulating translation: further characterization of mRNA discriminatory initiation factors.

Host and reovirus mRNAs compete with one another for translation in infected cells. Kinetic analysis has suggested that the site of competition is a message discriminatory initiation factor which must bind to the mRNA before it can interact with the 40S ribosomal subunit. The present communication describes an in vitro assay which can detect message discriminatory activities.

Comparative actions of phenytoin and other anticonvulsant drugs on potassium- and veratridine-stimulated calcium uptake in synaptosomes.

The actions of phenytoin and several anticonvulsants drugs on veratridine-stimulated (Na-dependent) and K-stimulated (Na-independent) Ca uptake were studied in isolated nerve terminals (synaptosomes) from rat cerebral cortex. Phenytoin inhibits both veratridine- and K-induced Ca uptake but is more potent against veratridine. Inhibition of veratridine-stimulated Ca uptake by phenytoin appears to be a competitive relationship, whereas the interaction between K and phenytoin is noncompetitive.

In vitro colony assay for a new class of megakaryocyte precursor: colony-forming unit megakaryocyte (CFU-M).

A plasma culture system has been used successfully to grow and quantitate megakaryocyte colonies from mouse bone marrow following their staining for acetylcholinesterase activity in situ. Colonies averaging about six acetylcholinesterase-positive cells appear with a peak incidence after 4 days in culture with a plating efficiency of one colony formed for every 10(4) nucleated cells plated.