Quantitative RT-PCR analysis of pacifastin-related precursor transcripts during the reproductive cycle of solitarious and gregarious desert locusts.

In locusts, little is known about the physiological and biochemical mechanisms regulating complex processes, such as reproduction and phase transition. The pacifastin family constitutes a family of peptidic inhibitors of serine proteases that are considered to be important regulators of several physiological processes in arthropods. We have performed a detailed transcript profiling analysis of two pacifastin-related peptide precursors, SGPP-2 and SGPP-4, during the reproductive cycle of adult desert locusts (Schistocerca gregaria). This quantitative real-time (RT)-PCR analysis revealed a temporal regulation of both transcripts, which is paralleled by several events that occur during the reproductive cycle of adult locusts. The observed temporal transcript profiles display a strong tissue-, gender- and phase-dependence. In addition, a partial regregarization experiment suggests that both transcript levels are regulated during phase transition and can be employed as molecular markers of the gregarization process.

Magnetic resonance imaging after seizures in patients with an ischemic stroke.

BACKGROUND: Seizures related to ischemic strokes are harmful. Their pathogenesis is not very well understood. The present study investigates whether diffusion-weighted imaging (DWI) can detect if those seizures are due to recurrent infarction or responsible for secondary ischemic changes. PATIENTS AND METHODS: DWI was obtained within 8 days in 60 patients with seizures (7 early and 53 late onset) related to an ischemic stroke. RESULTS: In 30 patients, positive DWI with decreased apparent diffusion coefficient was found. In 11 patients with late-onset seizures, the DWI showed a positive rim, surrounding the old infarct, while in 12 patients a large positive zone corresponding to a new infarct was observed. All 7 patients with early-onset seizures had positive DWI corresponding to the establishing infarct. A large positive zone on DWI was mainly observed in cardioembolic stroke. CONCLUSIONS: The positive rim around the old infarct can be considered as cytotoxic edema. Recurrent acute infarction, mainly of cardioembolic origin, seems to be a significant cause of so-called late-onset seizures, making the subdivision into early- and late-onset seizures arbitrary.

EEG findings after a cerebral territorial infarct in patients who develop early- and late-onset seizures.

BACKGROUND: EEG findings are generally not considered to be very helpful for the diagnosis of poststroke seizures. PURPOSE: This retrospective study investigates the EEG characteristics in patients who develop seizures after a cerebral territorial infarct. PATIENTS AND METHODS: The study population consisted of 110 patients with seizures after a cerebral territorial infarct (12 with early- and 98 with late-onset seizures) and 275 without. All 110 patients had an interictal EEG after their first seizure. The EEG patterns after the stroke were compared between those available from 69 patients who developed seizures and those from 275 who did not. Also the EEG patterns after the seizure (n = 110) were compared to those in the poststroke group without subsequent seizures. RESULTS: Periodic lateralized epileptic discharges (PLEDs) on the EEG after stroke were only found in 5.8% of the patients with early- and late-onset seizures. They were absent in the stroke group without seizures. Frontal intermittent rhythmic delta activities (FIRDAs) were observed in 24.6% of the seizure group, compared to 1.1% in the control group. Diffuse slowing occurred also significantly more often in the former (21.7%) compared to the latter group (5.1%). Normal EEG findings were seen in 53.8% of the stroke patients without seizures, compared to 8.5% in those with seizures. The incidence of focal slowing was the same in both groups. Similar findings were observed when comparing the EEG patterns of the patients after the first poststroke seizure to those of the stroke group without subsequent seizures. In patients with early-onset seizures, PLEDs or FIRDAs were present in 25% each. FIRDAs and diffuse slowing were significantly more frequently observed on the poststroke EEGs of patients who developed late-onset seizures. CONCLUSIONS: FIRDAs, PLEDs and diffuse slowing are the most frequent EEG findings in patients with early-onset seizures. Patients with FIRDAs and diffuse slowing on the poststroke EEG have a high risk to develop late-onset seizures, while the chance is reduced in those with normal EEG findings.

Computed tomographic changes of the brain and clinical outcome of patients with seizures and epilepsy after an ischaemic hemispheric stroke.

It is not well established whether seizures and epilepsy after an ischaemic stroke increase the disability of patients. Seventy-two patients with delayed seizures after a hemispheric infarct (37 with a single seizure and 35 with epilepsy) were included in the study. The modified Rankin scale was used to compare disability of the patients at 1 month after stroke and at 2 weeks after single or the last seizure, in case of epilepsy. The size of the X-ray hypoattenuation zone was compared on computed tomographic (CT) scans, performed in the weeks after the stroke and 1 week after single or repeated seizures. Lesion size was determined by superimposing the CT slices on digital cerebral vascular maps, on which the contours of the infarct area were delineated. The extent of the infarcts was expressed as the percentage fraction of the total surface area of the cerebral hemisphere. Groups with a single seizure and with epilepsy were mutually compared. Infarcts predominated in the parieto-temporal cortical regions. In the overall group the median Rankin score worsened significantly after seizures. The average size of the X-ray hypoattenuation zone was also significantly increased on the CT scans after the seizures, compared with those after stroke, without clear evidence of recent infarction. Mutual comparison of patients with a single seizure episode and of those with epilepsy showed only a trend of more severe disability and of increase in lesion size in the post-stroke epilepsy group. Delayed seizures and epilepsy after ischaemic stroke are accompanied by an increase in lesion size on CT and by worsening of the disability of the patients. This study does not allow to determine whether this is due to stroke recurrence or due to additional damage as a result of the seizures themselves.

Quantitative real-time RT-PCR analysis in desert locusts reveals phase dependent differences in neuroparsin transcript levels.

In different parts of the world, locust swarms cause severe ecological and economic damage. However, the physiological mechanisms underlying this gregarization process remain elusive. In this study, we present a detailed quantitative analysis of two neuroparsin precursor (Scg-NPP1 and Scg-NPP2) transcripts in the brain, fat body, gut, gonads and accessory glands of male and female, gregarious and solitarious desert locusts (Schistocerca gregaria). These transcripts are generally more abundant in solitarious than in gregarious animals. In contrast to their gregarious congeners, solitarious locusts contain detectable Scg-NPP1 and Scg-NPP2 transcript levels in the fat body. Moreover, our data reveal temporal changes of neuroparsin mRNA levels in the brains and fat bodies of adult isolated-reared locusts. This paper provides the first scientific evidence for phase-dependent transcriptional regulation of neuropeptide hormone encoding genes.

Neuroendocrinological and molecular aspects of insect reproduction.

This review summarizes recent advances and novel concepts in the area of insect reproductive neuroendocrinology. The role of 'classic' hormones, such as ecdysteroids and juvenoids, to control reproduction is well documented in a large variety of insect species. In adult gonads, ecdysteroids appear to induce a cascade of transcription factors, many of which also occur during the larval molting response. Recent molecular and functional data have created opportunities to study an additional level of regulation, that of neuropeptides, growth factors and their respective receptors. As a result, many homologs of factors playing a role in vertebrate reproductive physiology have been discovered in insects. This review highlights several neuropeptides controlling the biosynthesis and release of the 'classic' insect hormones, as well as various peptides and biogenic amines that regulate behavioural aspects of the reproduction process. In addition, hormone metabolizing enzymes and second messenger pathways are discussed with respect to their role in reproductive tissues. Finally, we speculate on future prospects for insect neuroendocrinological research as a consequence of the recent 'Genomics Revolution'.

cDNA cloning and transcript distribution of two novel members of the neuroparsin family in the desert locust, Schistocerca gregaria.

This study describes the identification and distribution of two novel neuroparsin precursor transcripts (Scg-NPP3/Scg-NPP4) in the desert locust, Schistocerca gregaria. Unlike Scg-NPP1 and Scg-NPP2, both transcripts were not only detected in the brain, but also in various other tissues, such as fat body, ventral nerve cord, testis and male accessory glands. Northern analysis showed that the levels of these transcripts are regulated during larval development, as well as during moulting and reproductive cycles. A significant increase in both mRNAs was observed during the period that just precedes the initial sexual activity of adult females and males. In silico analysis of sequence databases revealed the existence of several other neuroparsin-like peptides in a variety of arthropod species, including crustaceans and chelicerates. Neuroparsins also display similarities with vertebrate IGFBP.

The value of radiographic film for the characterization of intensity-modulated beams.

In this paper the performance of radiographic film (KODAK X-Omat V) for analysing intensity-modulated (IM) beams in a plane at reference depth (5 cm for 6 MV, 10 cm for 18 MV) was investigated. The field size dependence of the film response was studied for small and medium field sizes. The dose rate dependence of the response and possible effects of fractionating the dose were assessed. In the end, profiles were measured for two clinically delivered IM beams, and the results were compared with diamond detector data. We found that the response of the radiographic film increases with field size, but for field sizes up to 15 x 15 cm the deviations remain within 3% for measurements with the films in a plane at reference depth. We found that the response of the films decreases with decreasing dose rate, and that the extent of this effect differs from film batch to film batch. For clinical IM beams the effect can amount to about 9% at the location of shielded organs at risk. Also, fractionating the dose reduces the net optical density, but this effect is normally small when assessing IM beams. In low-dose regions low-energy photons have an important contribution, resulting in a higher response at these positions. This may counteract the dose rate dependence of the response. In the high-dose regions of the two IM beams that were studied, the relative dose measurements with film are within 1% of those obtained with a diamond detector, when the results of three films are averaged. In shielded organs at risk the deviations can mount to about 3%, depending on the film batch. In conclusion, radiographic film is a suitable detector for characterizing IM beams in a plane at reference depth.

Cloning of two cDNAs encoding isoforms of a pacifastin-related precursor polypeptide in the desert locust, Schistocerca gregaria: analysis of stage- and tissue-dependent expression.

A novel serine protease inhibitor peptide family, designated as the 'pacifastin family', has recently been described in insects (locusts, lepidopterans) and crustaceans (crayfish). This study presents the cDNA cloning of two isoforms of SGPP-3, a novel pacifastin-related precursor in the desert locust, Schistocerca gregaria, which codes for three putative inhibitor peptides. The precursor isoforms differ at a single amino acid position in the third, C-terminal peptide. Northern blot analysis confirmed the presence of two different transcripts (0.75 and 0.90 kb). Both transcripts are most abundant in the fat body and appear to be strongly regulated during the moulting cycle. In addition, the amount of transcript proved to be strictly regulated in the ovaries during the female reproductive cycle.

cDNA cloning of two different serine protease inhibitor precursors in the migratory locust, Locusta migratoria.

Recently, a novel serine protease-inhibiting peptide family, designated as the 'pacifastin family', has been described in locusts and crayfish. All members of this family possess a characteristic cysteine-rich domain. The present study describes the cDNA cloning, sequencing and transcript distribution of two novel pacifastin-related peptide precursors in the migratory locust, Locusta migratoria. Only one of the encoded peptides (HI) was identified previously, whereas six others represent new members of the pacifastin family. Northern blot analysis showed that both precursor transcripts are present in adult locust fat body. These could not be detected in the midgut. Interestingly, an in silico data mining approach of the expressed sequence tags (EST) database revealed the existence of Manduca sexta and Bombyx mori cDNAs that display pronounced sequence similarities with these locust pacifastin-related transcripts.

cDNA cloning and transcript distribution of two different neuroparsin precursors in the desert locust, Schistocerca gregaria.

Neuroparsins were originally identified in locust corpus cardiacum extracts as folliculostatic or 'antigonadotropic' neuropeptides. This paper presents the cloning of two different neuroparsin precursor cDNAs from the brain of the desert locust, Schistocerca gregaria. The first transcript encodes the precursor (Scg-NPP1) of S. gregaria neuroparsin A and B, whereas the second codes for a novel neuroparsin-related peptide precursor (Scg-NPP2). Both precursors display significant sequence similarities with each other and with the Locusta migratoria neuroparsin (Lom-NPP) and Aedes aegypti ovary ecdysteroidogenic hormone (Aea-OEH1) precursors. Northern blot analysis revealed that these neuroparsin transcripts are present in larval and adult locust brains. Interestingly, the Scg-NPP2 mRNA content proved to be strongly regulated during the reproductive cycle in both adult males and females.

Gonadotropins in insects: an overview.

Control of gonad development in insects requires juvenile hormone, ecdysteroids, and a peptidic brain gonadotropin(s). Compared to vertebrates, the situation in insects with respect to the molecular structure of gonadotropins is far less uniform. Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) of vertebrates are glycoproteins that are synthezised in the hypothalamus and released from the anterior pituitary. They stimulate gonad development, the production of progesterone or of sex steroids (estrogens, androgens). None of the known insect gonadotropins is a glycoprotein, neither can they be grouped into a single peptide family. In Drosophila, two G-protein coupled receptors, structurally related to the mammalian glycoprotein hormone receptors, have been identified. Nothing is known about their natural ligands. The sex-steroids of insects are likely to be ecdysteroids (20E in females, E in males of some species). Some of the identified gonadotropins speed up vitellogenesis (locust OMP and some -PF/-RFamide peptides) or stimulate ecdysteroid production by the ovaries (locust-OMP and Aedes- OEH) or testis (testis ecdysiotropin of Lymantria). In flies, the only as yet identified gonadotropin is the cAMP-generating peptide of Neobellieria. The seeming absence of uniformity in gonadotropins in insects might be due to a multitude of factors that can stimulate ecdysteroid production and/or to the use of different bioassays. Arch.

A recognisable behavioural phenotype associated with terminal deletions of the short arm of chromosome 8.

We report the clinical findings in 5 patients with a terminal deletion of the short arm of chromosome 8. Mild developmental delay was constantly present, in association with microcephaly in 4 of 5 patients. Facial anomalies were mild or absent. A congenital heart defect was present in 3 patients: an atrioventricular septal defect (AVSD) in 2 and an atrial septal defect type II (ASDII) with pulmonary stenosis in one. A highly similar pattern of behavioural difficulties was present in the 3 older children (8-11 years), with outbursts of aggressiveness and destructive behaviour. Follow-up in one patient showed that at the age of 16 years, these behavioural problems had largely disappeared. This observation suggests that in addition to mental retardation, microcephaly, congenital heart defect (typically AVSD), a terminal deletion of chromosome 8p may be associated with a characteristic behavioural phenotype during childhood.

All-trans-retinoic acid: measurement of reference values in human serum by high performance liquid chromatography.

A quantitative determination of physiological levels of all-trans-retinoic acid (vitamin A acid) in human serum has been developed. A double-phase extraction of 3.5 ml of serum followed by a specific and sensitive high performance liquid chromatography procedure allowed measurement of levels down to 1 ng/ml. Serum concentrations in 37 fasting volunteers ranged from 2.7 to 4.2 ng/ml and fitted a normal-Gaussian distributional shape with a mean value of 3.5 ng/ml and SD of 0.4 ng/ml, as demonstrated by the Kolmogorov-Smirnov test.

Multidimensional liquid chromatography: a breakthrough in the assessment of physiological vitamin K levels.

Owing to the lack of sensitivity and/or selectivity of the existing chemical assays, vitamin K deficiency has always been diagnosed indirectly by measuring its effect on blood coagulation. We used our recently developed multidimensional liquid chromatographic assay for what is, to the best of our knowledge, the first systematic investigation of physiological vitamin K levels in human blood. It allowed the unequivocal demonstration of trans-phylloquinone (vitamin K1) and its quantification down to a level of 0.5 nanogram per milliliter of serum (ng/ml. In healthy adults, a mean serum concentration of 2.6 ng/ml was found, with a normal range of 0.9 to 7.8 ng/ml. These values apparently are distributed in a log-normal way.

Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation.

A temperature-sensitive mutant having a lethal mutation in the gene for the beta subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) exhibits an apparent 2- to 3-fold decrease in the rates of both beta and beta' subunit synthesis at the non-permissive temperature, relative to total protein. In contrast, a temperature-sensitive mutant with a lethal mutation in the gene encoding beta' has a 5- to 6-fold increase in the rates of beta and beta' synthesis at 42 degrees. These beta and beta' mutants also exhibit rapid degradation of both subunits at the high temperature.