Catecholamine and MHPG plasma levels,platelet MAO activity,and3H-imipramine binding in heroin and cocaine addicts

This work evaluated in a population of heroin and heroin plus cocaine human addicts:

1. Norepinephrine (NE), epinephrine (Epi), and 3-methoxy-4-hydroxyphenylglycol (MHPG) (the principal metabolite of brain NE) plasma levels;
2. Monoamine oxidase (MAO) activity; and
3. ³H-imipramine specific binding to the amine carrier in platelets.

NE plasma levels were significantly lower in the short-term heroin user groups (1–3 and 4–6 yr), a finding not observed in both the long-term heroin user (>6 yr) and heroin plus cocaine user (>6 yr) groups. Epi levels changed in a similar manner, except that a significant increase was noted in heroin plus cocaine abusers. Conversely, dopamine and MHPG plasma levels increased with the duration of heroin use, and even more with cocaine abuse. Platelet MAO activity increased in all groups. Specific³H-imipramine binding sites showed an increase after 3 yr of heroin abuse and in all heroin plus cocaine addicts. In conclusion, short-term use of heroin decreases NE or Epi release, but with prolonged use, a slow adpatation occurs. In contrast, cocaine inhibits the neuronal Epi uptake, even in a situation of long duration of abuse. Probably the amine levels additionally regulate the amine carrier, resulting in changes that show a different pattern from major depression. These drugs of abuse may also influence directly or indirectly related enzymatic systems.     

Photoperiodic time measurement in the spider mite Tetranychus urticae: A novel concept

To explain photoperiodic induction of diapause in the spider mite Tetranychus urticae a new theoretical model was developed which took into account both the hourglass and rhythmic elements shown to be present in the photoperiodic reaction of these mites. It is emphasized that photoperiodic induction is the result of time measurement as well as the summation and integration of a number of successive photoperiodic cycles: the model, therefore, consists of separate ‘clock’ and ‘counter’ mechanisms. In current views involvement of the circadian system in photoperiodism is interpreted in terms of the hypothesis that the photoperiodic clock itself is based on one or more circadian oscillators. Here a different approach has been chosen as regards the role of the circadian system in photoperiodism: the possibility, previously put forward by other authors, that some aspect of the photoperiodic induction mechanism other than the clock is controlled by the circadian system was investigated by assuming a circadian influence on the photoperiodic counter mechanism. The derivation of this ‘hourglass timer oscillator counter’ model of photoperiodic induction in T. urticae is described and its operation demonstrated on the basis of a number of diel and nondiel photoperiods, with and without light interruptions.     

Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.     

High conservation of sequences involved in cystic fibrosis mutations in five mammalian species

Several mutations have been identified in the first nucleocide binding fold (NBF) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We have analyzed the DNA sequences of exons 10 and 11 in five different mammalian species, marmoset, mouse, cow, pig, and sheep; the amino acid conservation studied for nine disease mutations; and two “benign” mutations. For exon 10,87% homology at the DNA level and 93.5% at the amino acid level were found for these species. For exon 11, the lowest homology (70%), as found in mouse and the highest in marmoset (93%), whereas the amino acid sequence conservation ranged from 82.5 to 100%. All codons involved in CF mutations are highly conserved throughout evolution.     

The search for south European cystic fibrosis mutations: identification of two new mutations, four variants, and intronic sequences

The major mutation in the cystic fibrosis (CF) gene is a 3-bp deletion (delta F508) in exon 10. About 50% of the CF chromosomes in Southern Europe carry this mutation, while other previously described mutations account for less than 4%. To identify other common mutations in CF patients from the Mediterranean area, we have sequenced, exon by exon, 16 chromosomes that did not show the delta F508 deletion from a selected panel of eight unrelated CF patients. We describe here one missense and one nonsense mutation, and four sequence polymorphisms. We have also found two previously reported mutations in three chromosomes. Overall, these mutations may account for about 20% of CF alleles in the Italian and Spanish populations. No other mutations were detected in 10 out of 16 CF chromosomes after analyzing about 90% of the coding region of the CF gene, and 39 out of 54 intron/exon boundaries. Therefore, about 26% of CF mutations remain to be identified. In addition we provide the intron/exon boundary sequences for exons 4 to 9. These results together with previously reported linkage data suggest that in the Mediterranean populations further mutations may lie in the promoter region, or in intron sequences not yet analyzed.     

The origin of the nascent blastocoele in preimplantation mouse embryos ultrastructural cytochemistry and effect of chloroquine

Summary Mouse morulae are known to undergo cavitation as soon as some external cells have entered the sixth cell cycle (Garbutt et al. 1987). Since the early cytological features of cavitation are still unclear, we undertook a careful ultrastructural analysis of late morulae-nascent blastocysts. In addition, since maturation of lysosomes might be involved in the first step of cavity formation, we focused our attention on these organelles by means of the cytochemical localization of trimetaphosphatase activity and by the study of the effects of chloroquine on precavitation embryos. Our results suggest that cavitation starts in a few external cells (presumably competent cells entering the sixth cell cycle), by the chloroquine-sensitive formation of degradative autophagic vacuoles engulfing lipid droplets and vacuoles containing osmiophilic material. These complex structures enlarge (as a result of lipid metabolism?) and so transform into intrablastomeric cavities which, by means of a membrane fusion process, very rapidly become extracellular cavities that coalesce. The abembryonic pole of the blastocyst is determined in this way. Moreover, we suggest that the juxtacoelic cytoplasmic processes covering the inner cell mass (ICM) cells, which are known to restrict the expression of their totipotency during early cavitation (Fleming et al. 1984), are the latest remnants of the walls of the growing intrablastomeric cavities.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Micro-buffy coats of whole blood: a method for the electron microscopic study of mononuclear cells

A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.