Nerve Growth Factor influences potassium movements in chick embryo dorsal root ganglionic cells

Recently we have shown that Nerve Growth Factor (NGF) influences the movement of Na⁺ across the membrane of chick embryo dorsal root ganglion (DRG) cells. When cell dissociates from 8-day embryonic chick DRG, equilibrated with ⁸⁶Rb⁺ (a K⁺ analog) in the presence of NGF, were transferred to NGF-free medium a marked loss of intracellular K⁺ occurred over several hours. The time course of K⁺ loss was similar to the time course of Na⁺ accumulation which occurs in the absence of NGF. NGF-deprived, K⁺-depleted DRG cells reaccumulated K⁺ within minutes of delayed NGF presentation, just as delayed NGF administration results in the rapid extrusion of Na⁺ from Na⁺-loaded cells. Restoration of K⁺ competence was dependent upon NGF concentration. The occurrence of this K⁺ response to exogenous NGF in other ganglionic preparations correlated with traditional responses to NGF in culture and previously observed Na⁺ responses. Neither the development nor the expression of the ionic defect (K⁺ depletion, Na⁺ filling) during NGF deprivation required the presence of both cations in the medium. NGF-dependent restoration of intracellular K⁺ in NGF-deprived chick DRG cells required the presence of intracellular Na⁺, and NGF-dependent extrusion of Na⁺ required extracellular K⁺. Thus NGF appears to influence the coupled (active) movements of Na⁺ and K⁺ across the membrane of its target cells, possibly by means of the classical Na⁺, K⁺-ATPase pump.     

An attempt to prevent or delay denervation changes in rat muscles

A number of drugs which are known to affect lysosomes and their enzyme activities were used in an attempt to inhibit or delay the onset of denervation changes in rat muscles. The following parameters were used: the occurrence of fibrillations in electromyographs; diameters of muscle fibers; acid phosphatase activity; acetylcholinesterase activity and distribution in end plates. Differences between denervated and non-denervated limbs were evaluated and compared in the different treatment groups. The various parameters were differently affected by the different drugs. Chloroquin, thiouracil and streptomycin appeared to be more effective than other treatments in the inhibition of denervation changes.     

Evaluation of ketanserin as a tocolytic agent in third trimester pregnant rats

Ketanserin, as a 5HT2 selective antagonist, was evaluated for its tocolytic effects in pregnant Charles River (CR) rats, at total doses of 1.5 and 3.5 mg/kg IU from day 22 to day 25 of pregnancy. After 18 hours of ethenyl estradiol induction, tocolytic evaluation was carried out by recording uterine activity. When compared to the control group, the administration of Ketanserin resulted in an apparent, nondose dependent suppression of uterine contractility.     

The microbiome of Brazilian mangrove sediments as revealed by metagenomics

Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H(2)S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments.     

Hydroxylation of p-substituted phenols by tyrosinase: further insight into the mechanism of tyrosinase activity

A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k(cat)(m) and the Michaelis constant, K(M)(m). Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group (δ) and σ(p)(+), enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form E(ox) (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant ρ of -1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of [Formula: see text] / [Formula: see text] against n (atom fractions of deuterium), where [Formula: see text] is the catalytic constant for a molar fraction of deuterium (n) and [Formula: see text] is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that only one of the proton transfer processes from the hydroxyl groups involved the catalytic cycle is responsible for the isotope effects. We suggest that this step is the proton transfer from the hydroxyl group of C-1 to the peroxide of the oxytyrosinase form (E(ox)). After the nucleophilic attack, the incorporation of the oxygen in the benzene ring occurs by means of an electrophilic aromatic substitution mechanism in which there is no isotopic effect.     

Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase

We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamineso-aminophenols>aromatic o-diamines.     

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. II. Contribution of the uni- and bimolecular activation routes

From the kinetic study carried out in part I of this series (preceding article) an analysis quantifying the relative contribution to the global process of the uni- and bimolecular routes has been carried out. This analysis suggests a way to predict the time course of the relative contribution as well as the effect on this relative weight of the initial zymogen, inhibitor and activating enzyme concentrations.     

Human RAD50 Deficiency in a Nijmegen Breakage Syndrome-like Disorder

The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, ‘bird-like’ face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell-cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD).     

High prevalence of theNBN gene mutation c.657-661del5 in Southeast Germany

Nijmegen breakage syndrome (NBS), a rare autosomal recessive chromosomal instability disorder, is caused by mutations in theNBN gene. Most patients known so far are of Slavic origin and carry the major founder mutation c.657-661del5. Due to an unexpectedly high incidence of NBS patients (homozygous for the c.657-661del5 mutation) in a Northeast Bavarian region in Southeast Germany, we estimated the prevalence of this mutation in this area and compared it to another German region. We found a high carrier frequency of 1/176 for the c.657-661del5 mutation among newborns in Northeast Bavaria, while the frequency of the mutation in Berlin was 1/990. We further studied families from a Slavic population isolate, the Sorbs, in the Lusatian region in Northeast Saxony, and revealed a prevalence of the c.657-661del5 mutation of 1/34. Whereas the Slavic origin of the Sorbs has been known, we attribute the surprisingly high frequencies of c.657-661del5 mutation in Bavaria (similar to frequencies of this mutation in various Eastern European countries) to a high percentage of people of Slavic origin in Northeast Bavaria.     

Cdc42-driven podosome formation in endothelial cells

Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.