Target-Dependent Regulation of Retinal Nicotinic Acetylcholine Receptor and Tubulin RNAs During Optic Nerve Regeneration in Goldfish

A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in alpha-tubulin RNAs (3 days), which preceded the increase in nicotinic acetylcholine receptor RNAs (10-15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4-6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Inability of IL-2 and IL-10 to counteract B cell clonal deletion.

The B cell antigen receptor (BCR) delivers inhibitory signals in nascent B cells leading to the establishment of tolerance via clonal deletion or clonal anergy depending upon the type of antigen to which the B cells are exposed. In previous work, it has been demonstrated that activated Th2 cells, as well as some recombinant lymphokines, prevent the inhibition of growth and subsequent cell death induced through the BCR in model B cell lymphomas. Herein, we extend this work to another Th2 lymphokine, IL-10, that in contrast to IL-4 does not interfere with the deletion promoted by IgM crosslinking. The effect of individual lymphokines has also begun to be analyzed in a transgenic model of B cell clonal deletion. To this end, we have administered a recombinant vaccinia virus producing human IL-2 to mice expressing an autoreactive H-2Kk,b-specific transgenic IgMk and found that IL-2 does not abrogate B cell deletion in vivo.     

Germination response to temperature,salinity, light and depth of sowing of ten tropical dune species

This study describes the germination responses of ten tropical dune species to several factors to which their seeds are exposed in the field. Species studied were: three sand dune endemics (Amaranthus greggii, Palafoxia lindenii, and Trachypogon gouini), three pantropical coastal species (Sesuvium portulacastrum, Sporobolus virginicus and Ipomoea stolonifera) and four cosmopolitan grasses also found inland (Panicum repens, Panicum maximum, Pappophorum vaginatum and Andropogon glomeratus). Six species germinated when exposed to different constant temperatures. Four required temperature fluctuation (S. portulacastrum, P. lindenii, S. virginicus, P. repens). Light promoted germination of three species (S. Portulacastrum, P. vaginatum, P. lindenii). Species varied in their degree of sensitivity to salinity. Seeds of T. gouini, I. stolonifera, S. portulacastrum, P. repens, P. Maximum and P. vaginatum were able to germinate under some of the salinity concentrations. Not all species were able to recover after being transferred to distilled water. Seedling emergence was inhibited when seeds were buried. This response was related to depth and to seed size. S. portulacastrum and S. virginicus were the most affected. Nitrates only affected germination response of two species. Seed age promoted germination under a wider range of conditions. P. lindenii showed very heterogeneous responses depending on seed cohort. No dormancy mechanisms were found, other than a thermoperiod and/or light requirement for some of the species. Cosmopolitan grasses tolerated both sand burial and salinity, although the endemics were the most successful in emerging from sand burial; coastal pantropicals were very tolerant to high saline concentrations and recovered completely.     

Use of triple stain technique for simultaneous assessment of vitality and acrosomal status in boar spermatozoa

The object of this study was to adapt the triple stain technique to diluted and incubated boar spermatozoa. Freshly ejaculated semen was resuspended in MR-A diluent to contain 3x10(7) cells/ml (diluted spermatozoa) and was subsequently capacitated (incubated spermatozoa). Experiments were conducted to show the conditions required for optimal staining quality and validation of triple stain technique. The most satisfactory staining solutions for diluted spermatozoa were 2% Trypan blue at 37 degrees C for 15 minutes, 0.8% Bismarck brown in 30% ethyl alcohol (pH 2.8) at 40 degrees C for 10 minutes and 0.8% rose Bengal in 0.1 M of Tris (pH 4.3) at 21 degrees C for 20 minutes. Satisfactory results were obtained for incubated spermatozoa with rose Bengal when the staining time was 10 minutes. Triple stain technique seemed to be a useful method for the simultaneous assessment of sperm vitality and acrosomal status; consequently, it should be valuable tool, for use in porcine in vitro fertilization systems.     

Analysis of centromere size in human chromosomes 1, 9, 15, and 16 by electron microscopy.

Human chromosomes were treated with 5-azacytidine and analyzed by whole-mount electron microscopy. This base analogue produces undercondensation of heterochromatin and separation of the centromere from the bulk of pericentromeric heterochromatin in chromosomes 1, 9, 15, and 16, which allows clear delimitation of the centromere regions. A quantitative analysis of centromeres showed that chromosomes 1, 9, and 16 have centromeres of different size. The centromere of chromosome 15 is similar in size to that of chromosome 9 and different from those of chromosomes 1 and 16. No interindividual variation for centromere size was found. A positive correlation between centromere and chromosome size was found for the chromosomes analyzed.     

Members of the alpha-amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma

We have identified the major antigens or IgE binding components from wheat flour. Thirty-five sera from patients with baker's asthma were used to analyze the reaction with wheat salt-soluble proteins. We found a 15 kDa SDS-PAGE band which reacted with all sera tested. Purified members of the alpha-amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two-dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low-molecular-weight allergens.     

Genetic stability of the antagonistic character ofEnterococcus faecalis ssp.liquefaciens and the detection of a new inhibitory bacteriocin-like substance

The inhibitory capacity of strain S-48 ofEnterococcus faecalis ssp.liquefaciens was studied. The strain produces a broad-spectrum peptide antibiotic (AS-48) that has been characterized elsewhere. The isolation of mutants from S-48 after mutagenic treatment revealed another inhibitory substance which remained masked in the wild strain. The protein nature and restricted spectrum of this substance points to its being a bacteriocin (Bc-48).     

Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi

A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.     

Computer simulation of hydrodynamic properties of semiflexible macromolecules: randomly broken chains, wormlike chains, and analysis of properties of DNA

The translational and rotational diffusion coefficients and the intrinsic viscosity of semiflexible, randomly broken, and wormlike chains have been obtained by Monte Carlo simulation in the context of the rigid-body treatment. Both approximate and rigorous rigid-body hydrodynamics are used, so that the error introduced by the approximate methods can be evaluated. A randomly broken chain and a wormlike chain having the same contour length and persistence length have the same radius of gyration but different values for any of the hydrodynamic properties. The two types of chains are compared in this regard. Considering that the cross section of the chain is represented by a cylinder better than by a string of spheres, we devise a cylindrical correction to be applied to the results simulated for chains of beads. Application is made to the analysis of experimental data for the translational and rotational coefficients of DNA fragments with up to 10(3) base pairs, obtaining the persistence length for each model. The values for the wormlike chain agree well with model-independent values obtained from radii of gyration and with other literature data at varying ionic strength. The randomly broken chain is equally able to reproduce the experimental length dependence of the properties, but the resulting persistence length may be too high.