S1R agonist modulates rat platelet eicosanoid synthesis and aggregation.

Sigma-1 receptor (S1R) is detected in different cell types and can regulate intracellular signaling pathways. S1R plays a role in the pathomechanism of diseases and the regulation of neurotransmitters. Fluvoxamine can bind to S1R and reduce the serotonin uptake of neurons and platelets. We therefore hypothesized that platelets express S1R, which can modify platelet function. The expression of the SIGMAR1 gene in rat platelets was examined with a reverse transcription polymerase chain reaction and a quantitative polymerase chain reaction. The receptor was also visualized by immunostaining and confocal laser scanning microscopy. The effect of S1R agonist PRE-084 on the eicosanoid synthesis of isolated rat platelets and ADP- and AA-induced platelet aggregation was examined. S1R was detected in rat platelets both at gene and protein levels. Pretreatment with PRE-084 of resting platelets induced elevation of eicosanoid synthesis. The rate of elevation in thromboxane B2 and prostaglandin D2 synthesis was similar, but the production of prostaglandin E2 was higher. The concentration-response curve showed a sigmoidal form. The most effective concentration of the agonist was 2 µM. PRE-084 increased the quantity of cyclooxygenase-1 as detected by ELISA. PRE-084 also elevated the ADP- and AA-induced platelet aggregation. S1R of platelets might regulate physiological or pathological functions.

Linking wood anatomy with growth vigour and susceptibility to alternate bearing in composite apple and pear trees.

Excess vegetative growth and irregular fruit-bearing are often undesirable in horticultural practice. However, the biological mechanisms underlying these traits in fruit trees are not fully understood. Here, we tested if growth vigour and susceptibility of apple and pear trees to alternate fruit-bearing are associated with vascular anatomy. We examined anatomical traits related to water transport and nutrient storage in young woody shoots and roots of 15 different scion/rootstock cultivars of apple and pear trees. In addition, soil and leaf water potentials were measured across a drought period. We found a positive correlation between the mean vessel diameter of roots and the annual shoot length. Vigorously growing trees also maintained less negative midday leaf water potential during drought. Furthermore, we observed a close negative correlation between the proportions of total parenchyma in the shoots and the alternate bearing index. Based on anatomical proxies, our results suggest that xylem transport efficiency of rootstocks is linked to growth vigour of both apple and pear trees, while limited carbohydrate storage capacity of scions may be associated with increased susceptibility to alternate bearing. These findings can be useful for the breeding of new cultivars of commercially important fruit trees.

Circulating P-Selectin Glycoprotein Ligand 1 and P-Selectin Levels in Obstructive Sleep Apnea Patients.

PURPOSE: Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia which induces inflammation in blood vessels leading to the development of cardiovascular comorbidities. Several studies implicated the role of P-selectin in vascular inflammation of OSA. P-selectin glycoprotein ligand 1 (PSGL-1) is the main activator for P-selectin and is involved in immune cell trafficking. However, PSGL-1 has not been analyzed in OSA. The aim of the study was to investigate plasma PSGL-1 and P-selectin levels to have a deeper understanding on their interaction in obstructive sleep apnea. METHODS: Fifty-one untreated patients with OSA and 42 non-OSA controls were recruited. Plasma PSGL-1 levels were determined in evening and morning samples, P-selectin levels were analyzed in morning samples using commercially available ELISA kits. Polysomnography was performed in all participants. OSA was defined by an apnea-hypopnea index ≥ 5/h. RESULTS: PSGL-1 levels did not differ between controls and OSA patients either in the evening or in the morning. Although, there was no difference between controls (16.9/6.8-40.8 ng/ml) and patients with OSA (19.6/8.4-56.8, p = 0.24), patients with severe OSA had increased plasma P-selectin levels (25.6/8.4-56.8 ng/ml) compared to mild OSA patients (14.1/8.5-35.3 ng/ml, p = 0.006) and controls (p = 0.03). CONCLUSIONS: P-selectin expression relates to disease severity suggesting a pathophysiological role in endothelial cell activation. PSGL-1 levels are unaltered in OSA, suggesting an alternative activation pathway for P-selectin in OSA.

Up- and downregulation of esr20, an ecdysteroid-regulated gene expressed in the tracheae of Manduca sexta.

Investigations of ecdysteroid-regulated gene cascades in Drosophila have shown that characteristics of downstream genes in such cascades include their repression by high ecdysteroid levels, their expression at low hormone levels, and the dependence of their expression on protein synthesis. In an earlier study, we identified a gene, esr20, which is expressed in the tracheae of the tobacco hornworm, Manduca sexta, prior to larval and pupal ecdyses. Initial characterization of the expression of esr20 suggested that it had the above characteristics of a downstream gene in an ecdysteroid-regulated cascade. The present study shows that, unlike the downstream genes in Drosophila, the expression of esr20 in tracheae cannot be induced by changes in the ecdysteroid levels alone. We present evidence which suggests that a decline in ecdysteroid is necessary but not sufficient for expression. Soon after pupal ecdysis the level of the esr20 transcript drops fourfold, and by 24 h after ecdysis the transcript is undetectable. Evidence is presented which suggests that this decline in transcript levels requires protein synthesis and appears to result from a decline in the stability of the transcript.

Expression of a developmentally regulated gene, Mng10, in identified neurosecretory cells in the CNS of Manduca sexta.

We are interested in the molecular events underlying the development of the nervous system of Manduca sexta during the final 24 h of the pupal molt. In this article we describe a gene, Mng10, that is expressed in the abdominal nervous system of M. sexta and is developmentally regulated over this 24-h period. In situ hybridization analysis shows that the transcript is localized predominantly to a single pair of uniquely identifiable neurosecretory neurons, the NS-L1 cells in the abdominal ganglia. Mng10 is a single copy gene encoding a 229 amino acid protein with a predicted molecular mass of 26 kDa. At the amino acid level the protein shows 34% identity to the yeast transcription unit, Yer082. Northern blot analysis shows that the transcript of Mng10 is very rare, comprising about 0.001% of the poly (A)+ RNA from the CNS and is detectable at 4 h but not 24 h prior to pupal ecdysis. One of the physiological events that develops over the final 24 h of the pupal molt is the ability of the nervous system to respond to the neuropeptide eclosion hormone. In this context, it is interesting to note that the NS-L1 cells are members of the group of 50 neurons that show increased cGMP immunoreactivity when the nervous system is exposed to the neuropeptide eclosion hormone.

Comparison of the expression patterns of five developmentally regulated genes in Manduca sexta and their regulation by 20-hydroxyecdysone in vitro.

A previous study, using subtractive hybridization, identified five genes (esr16, esr20, Mng10, Mng14 and tps9) whose transcripts were up-regulated prior to metamorphosis in Manduca sexta nervous tissue. The developmental time points chosen for subtraction suggested that expression could be coordinately regulated and should be negatively regulated by the steroid hormones, the ecdysteroids. In the present paper, we present an analysis of the expression patterns of these five genes, using reverse transcription-polymerase chain reaction (RT-PCR), at various times during development and assess the effects of 20-hydroxyecdysone and cycloheximide on their expression in vitro. This analysis revealed that with the exception of esr20 all transcripts were detectable at all times and that all but one of the genes were up-regulated in vivo and in vitro in the absence or in the presence of low levels of the ecdysteroids. In the absence of ecdysteroids, cycloheximide blocked the accumulation of only two transcripts, esr16 and esr20. These results reveal a much more complex pattern of gene expression in the central nervous system prior to metamorphosis than previously imagined.

Subtractive hybridization strategy using paramagnetic oligo(dT) beads and PCR.

Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)+ RNA pool to paramagnetic Dynabeads Oligo (dT)25. After hybridization with target cDNA, the molecules common to both pools are removed. The subtracted cDNA is then amplified with PCR and used for library screening. Using this method, we have identified four cDNA clones that represent developmentally regulated transcripts in the central nervous system of the tobacco hornworm Manduca sexta. All four transcripts are of low abundance, comprising only 0.001%-0.5% of the poly(A)+ RNA pool.

Identification of a developmentally regulated gene, esr16, in the tracheal epithelium of Manduca sexta, with homology to a protein from human epididymis.

Events related to both ecdysis and metamorphosis require the expression of a new set of genes, the majority of which are regulated by the changes in ecdysteroid levels. We have initiated studies to identify genes whose expression is up-regulated between 24 and 4 h before pupal ecdysis in Manduca sexta. In this paper we report the partial characterization of one such gene, esr16. The transcript of esr16 is detected by Northern blot analysis in nervous tissue, muscle and trachea isolated from animals 4 h before, but not 24 h before pupal ecdysis. In situ hybridization showed that the transcript was expressed in epithelial cells of the large tracheae surrounding the nervous system and muscle. Sequence analysis suggested that the gene encoded a secreted protein with 35% identity to HE1, a human epididymal-specific gene.

Maturation of the GFAP-immunopositive glial network is delayed in the olfactory bulbs of rats subjected to early, low-dose X-irradiation.

Young rats were subjected to low-level, repeated doses of x-irradiation from a Co60 source and examined for the presence and arrangement of their GFAP-immunopositive system at the light and electron microscopic levels. In 2-week-old control rats, labelled cells were already distributed homogeneously throughout the bulb, while in the experimental animals, a (newborn- and early postnatal-type) radial arrangement of immunoreactive fibres continued to persist even until the end of the fourth postnatal week. This delay in the maturation of the GFAP-immunopositive glial system may contribute to the hindered development of the bulbar neurones, neuronal connections, and field potentials, as found in earlier studies.

Isolation and partial characterization of a gene from trachea of Manduca sexta that requires and is negatively regulated by ecdysteroids.

The molting cycle in insects is regulated by the fluctuating levels of ecdysteroids. Twenty-four hours prior to the pupal molt in Manduca sexta steroid titers are relatively high and they fall to very low levels at 4 hr before the molt. Several physiological events accompanying molts have been shown to require the rise and then the subsequent decline of ecdysteroid levels. These events also require the expression of a new set of genes. We used subtractive hybridization to isolate genes that are expressed 4 hr, but not 24 hr, prior to the pupal molt in the nervous system of Manduca. These genes are expected to play a role either in ecdysis behavior or in the development of pupal/adult structures. The first such gene we have identified, esr20 (EcdySteroid Regulated) encodes a 20-kDa protein. The deduced amino acid sequence of the protein shows similarity to acidic domains in three urelated proteins, chick nucleolin, rat heavy-molecular-weight neurofilament, and the Drosophila steroid orphan receptor, knirps-related. The similarity among the four proteins appears to be in regions implicated in protein-protein interactions. The accumulation of esr20 mRNA starts 16 hr prior to the pupal molt and abruptly stops at ecdysis. Expression of esr20 was localized to tracheal epithelial cells within all tissue types, and the transcript was present prior to the larval, pupal, and adult molts only after the molting peak of ecdysteroids had declined. Its expression was inhibited in the presence of ecdysteroids in vitro and required protein synthesis.

Low-dose X-irradiation reduces neuronal and glial cell populations in the juxtaglomerular region of rat olfactory bulb.

Rats were subjected to fragmented, low-dose, whole body X-ray irradiation from last prenatal to third postnatal week of life. Consequences of the irradiation were examined at the cellular and synaptic level and evaluated by light- and electron microscopy. The fate of several cell types of the glomerular layer was assessed by semi-quantitative morphometry. All known neuronal and glial cell subtypes and varieties of synaptic connections of this layer were spared. X-irradiation, however, had a marked influence on the composition of the juxtaglomerular cell population even at the youngest examined, two week old rat pups and this impact still persisted, without recovery, at six weeks of age. Density (and so, absolute number) of the small periglomerular neurones and astrocytes was depressed to 52-55% and 32-48% of control, respectively, while the density of the superficial tufted cells and the rest of glial cells remained at the control level. These results suggest that the small periglomerular neurones may primarily be responsible for the shrinkage of this layer. This means, in the same time that, selective depression of the development of a superficial interneuron (in addition to the deeply situated granule cells) could be achieved.

Ageing and problems in differential diagnosis of psychoorganic syndromes.

Depressive syndrome was investigated in 132 psychiatric and neurologic patients by Zung's self-rating depression scale. The dexamethasone suppression test (DST) was carried out in 62 patients. Visual evoked potentials (VEP) (stimulation with flash (F-VEP) and pattern (P-VEP)), serum levels of LH, FSH, prolactin, ACTH, estradiol and cortisol were measured in 15 female patients between 85 and 94 years of age. Abnormal DST results indicated depression rather than dementia. P-VEP latencies correlated with cognitive symptoms, while F-VEP latencies correlated with affective symptoms. Gonadotropin levels were lowered in senility in comparison with the climacteric period of life. Depressive, anxious, cognitive, anorexic, sexual and disturbed diurnal mood changes syndromes could be separated by factor analysis. The severity of the cognitive depressive syndrome correlated negatively with estradiol serum levels. The total scores of Zung's self-rating depression scales correlated positively with ACTH and negatively with FSH serum levels.

Early X-irradiation of rats. 5. Influence of irradiation on the development of extracellular field potentials evoked by antidromic stimulation.

Rat pups were treated by low, fragmented and repeated doses of X-rays, from late prenatal days until the end of the third postnatal week. Extracellular field potentials, evoked by antidromic stimulation of the lateral olfactory tract were recorded in different layers of the olfactory bulb at 1, 2, 3 and 6 weeks of age, from control (non-irradiated) and experimental rats. Development of the field potentials was analysed in both groups of animals. In controls, the amplitude of responses was gradually increasing along with age while the latency of peaks decreased; inhibitory waves were tuned even after the third postnatal week. When compared to controls at similar ages, irradiated rats had smaller peak amplitude of responses and the shortening of the response latency was delayed. In addition, a new, late-appearing excitatory wave component was observed in the granule cell layer by the sixth week. The effect of irradiation on field potentials of the olfactory bulb is discussed in light of the marked reduction of the inhibition in the local neuronal assembly, which is also indicated by the depressed development of its structural and neurochemical correlates.

Induction of multinucleated giant cell formation from human blood-derived monocytes by phorbol myristate acetate in in vitro culture.

Multinucleated giant cells (MGC) of mononuclear phagocyte origin occur in different tissues in various inflammatory states and pathological conditions. Although MGC are believed to be derived from monocyte-derived macrophages by fusion, their mechanism of formation is not known. In this study, we investigated the role of PMA, a protein kinase C activator, in the induction and formation of MGC from blood monocyte-derived macrophages in in vitro culture. The addition of PMA (1 x 10(-9) to 8 x 10(-8) M) to 3-wk-old cultures of blood-derived monocytes induces cell fusion with a 30% to 80% fusion rate. Moreover, IFN-gamma-treated blood-derived monocyte cultures showed an additional enhancement of fusion rate with the addition of PMA. 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a protein kinase inhibitor, inhibited the formation of macrophage-derived giant cells when added before phorbol ester and IFN-gamma. These findings suggest that protein kinase C may play an important role in the formation of macrophage-derived MGC.

Decomposition of N-(2-chloroethyl)-N-nitrosocarbamoyl amino acid amides.

The chemical decomposition of N-(2-chloroethyl)-N-nitrosocarbamoyl (Q(NO] prolinamide and valinamide were studied under physiological conditions. The volatile products were identified with GC. Q(NO)-Pro-NH2 gave twice the amount of ethylene glycol and only one-fifth of the 2-chloroethanol produced by Q(NO)-Val-NH2 or BCNU, pointing to different pathways of their decomposition. The carbamoylating activity was also investigated in the presence of cyclohexylamine, and it was found to lead mainly to intramolecular carbamoylation with the formation of hydantoin derivatives.

ECG conduction disturbance in the first-degree relatives of children with ventricular septal defect.

A detailed cardiological examination was made in the first-degree relatives of 94 index patients with ventricular septal defect and in 94 matched control cases. Incomplete or suspect right bundle branch blocks were found in 18.3% and in 4.1%, respectively, of first-degree relatives of index and control groups. The difference is highly significant. These intraventricular conduction disturbances may be subthreshold signs of septal defects.

Two family studies of children with ventricular septal defect.

All first-degree relatives of 81 index patients with isolated ventricular septal defects were examined cardiologically in sample one. The congenital abnormalities in first-degree relatives of 296 index patients affected ventricular septal defects were studied by questionnaire in sample two. (The relatives reported as having congenital cardiovascular malformations were checked). Ventricular septal defects were found in 3.3% and 1.45% of sibs in samples one and two, respectively. The heritability of isolated VSD was 0.57 +/- 0.22. The familial clustering fitted the multifactorial threshold model well. Other congenital cardiovascular malformations were somewhat higher in first-degree relatives of index patients (1.6% in sample one and 1.2% in sample two) than their expected rates. The occurrence of other congenital abnormalities, however, does not exceed the prevalence at birth in the population.