Home alone: Social functioning as a transdiagnostic marker of mental health in youth, exploring retrospective and daily life measurements.

PURPOSE: Early detection and intervention of mental health problems in youth are topical given that mental disorders often start early in life. Young people with emerging mental disorders however, often present with non-specific, fluctuating symptoms. Recent reports indicate a decline in social functioning (SF) as an early sign of specific emerging mental disorders such as depression or anxiety, making SF a favorable transdiagnostic approach for earlier detection and intervention. Our aim was to investigate the value of SF in relation to transdiagnostic symptoms, and as a predictor of psychopathology over time, while exploring traditional retrospective versus innovative daily diary measurements of SF in youth. METHOD: Participants (N = 75) were 16-25 years of age and presented early stage psychiatric symptomatology. Psychiatric symptoms, including anxiety and depression, as well as SF -both in retrospect and in daily life- were assessed at two time points and analyzed cross-sectionally and longitudinally. RESULTS: A significant and negative association between SF and all psychiatric symptoms was found, and SF was a significant predictor of change in general psychiatric symptoms over time. Results were only significant when SF was measured traditionally retrospective. CONCLUSION: This study confirms a distinct relation between SF and transdiagnostic psychiatric symptoms in youth, even in a (sub)clinical population, and points towards SF as a predictor of transdiagnostic psychiatric symptoms. Further research is needed to learn more about the added value of daily life versus retrospective measurements.

From laboratory to life: associating brain reward processing with real-life motivated behaviour and symptoms of depression in non-help-seeking young adults.

BACKGROUND: Depression has been associated with abnormalities in neural underpinnings of Reward Learning (RL). However, inconsistencies have emerged, possibly owing to medication effects. Additionally, it remains unclear how neural RL signals relate to real-life behaviour. The current study, therefore, examined neural RL signals in young, mildly to moderately depressed - but non-help-seeking and unmedicated - individuals and how these signals are associated with depressive symptoms and real-life motivated behaviour. METHODS: Individuals with symptoms along the depression continuum (n = 87) were recruited from the community. They performed an RL task during functional Magnetic Resonance Imaging and were assessed with the Experience Sampling Method (ESM), completing short questionnaires on emotions and behaviours up to 10 times/day for 15 days. Q-learning model-derived Reward Prediction Errors (RPEs) were examined in striatal areas, and subsequently associated with depressive symptoms and an ESM measure capturing (non-linearly) how anticipation of reward experience corresponds to actual reward experience later on. RESULTS: Significant RPE signals were found in the striatum, insula, amygdala, hippocampus, frontal and occipital cortices. Region-of-interest analyses revealed a significant association between RPE signals and (a) self-reported depressive symptoms in the right nucleus accumbens (b = -0.017, p = 0.006) and putamen (b = -0.013, p = .012); and (b) the quadratic ESM variable in the left (b = 0.010, p = .010) and right (b = 0.026, p = 0.011) nucleus accumbens and right putamen (b = 0.047, p < 0.001). CONCLUSIONS: Striatal RPE signals are disrupted along the depression continuum. Moreover, they are associated with reward-related behaviour in real-life, suggesting that real-life coupling of reward anticipation and engagement in rewarding activities might be a relevant target of psychological therapies for depression.

[Long Term Monitoring of Nutritional, Clinical Status and Quality of Life in Head and Neck Cancer Patients]. / Dlouhodobé sledování nutricního, klinického stavu a kvality zivota u nemocných s rakovinou hlavy a krku.

BACKGROUND: Malnutrition in head and neck cancer (HNC) patients decreases survival, quality of life (QOL) and oncological outcomes. The aim of the prospective three-year study was to compare QOL, clinical symptoms and variables (complications, survival and mortality rates in HNC patients). PATIENTS AND METHODS: A total of 726 patients aged 55 to 72 years with treatable HNC were included from January 2004 to December 2009; these patients were randomized to either group with PEG and enteral nutrition and nonPEG group with nutritional counselling according to nutritional care. We used EORTC questionnaires QOL C-30 and Head and neck module (HN-35) for measuring of QOL. The following variables due to expectable influence on QOL (demographic data, oncological data, nutritional screening, Clinical symptom score, Karnofsky performance status score, Charlson comorbidity index) were included. Monitoring was done five times in three years. RESULTS: In the first six months, we found decrease of weight and body mass index (BMI). After this critical time point and finish of oncological treatment, a marked difference in the development of patients treated with PEG. Negative factors influencing patients survival, QOL, clinical status were males aged > 63 years, hypopharyngeal cancer (stage III- IV), smoking, weight loss > 10%, BMI < 21 and disallowance of PEG. CONCLUSIONS: QOL is an essential factor for cancer patients. Our study showed that nutritional intervention with early enteral nutrition may improve QOL and survival in HNC patients. The PEG group better tolerated oncological treatment, had lower incidence of complications, shorter time to re-entry of permanent increase in weight, lower rate of rehospitalization and its shorter length. We found QOL questionnaires to be very important for better understanding and communication and a key instrument for improving solution of patients difficulties during their therapy in multidisciplinary approach.

Aductive laser iridoplasty and laser goniopuncture after non-perforating trabeculectomy.

INTRODUCTION: Successful non-perforating trabeculectomy (NPT) results in filtration of aqueous humor out of the anterior chamber and into a filtration bleb, without surgical excision of tissue from the anterior chamber angle, and without penetration into the anterior chamber. The complications of perforating trabeculectomy, due to early postoperative hypotony (shallow anterior chamber, hyphema, macular folds, suprachoroidal effusion, and ciliochoroidal hemorrhage) (3, 4, 5, 6, 7, 8, 9) are regarded by many surgeons as significant risks. Nonperforating surgery has been reported to reduce the incidence of early hypotony-related complications (10), because it has the advantage of creating gradual filtration of aqueous humor, through a thin trabeculodescemetic membrane (TDM), which markedly reduces postoperative complications seen after a conventional trabeculectomy (11), and also has been reported to provide better long-term intraocular pressure (IOP) control (12, 13). NPT is reported to be a procedure with a significant learning curve, sometimes necessitating conversion to perforating trabeculectomy, and requiring careful postoperative monitoring (14, 15, 16, 17). Zimmerman et al. reported filtration of aqueous humor under a filtering bleb, by resecting the roof of Schlemms canal and removing corneal stroma overlying the trabecular meshwork (18) Mermoud et al. reported filtration of aqueous humor under a filtering, bleb by unroofing Schlemms canal and removing corneal stroma overlying the trabecular meshwork as well Descemets membrane (19); he found that resistance across the TDM sometimes increased with time. When this resistance to aqueous humor outflow occurred, Mermoud found TDM resistance could be eliminated by performance of goniopuncture (ab interno Nd:YAG laser membranotomy via gonioprism), to enhance aqueous humor outflow into the filtration bleb. Failure to filter adequately through the TDM is a potential complication following NPT which can result in a rise in intraocular pressure (IOP). In this paper we examine the effectiveness of adjunctive Nd:YAG laser goniopuncture (YGP) in patients who underwent NPT, to reduce post-operative IOP rise, secondary to scarring at or poor aqueous outflow through the TDM. Iris prolapse (IP) is another potential complication following NPT which can result in a rise in IOP. In this paper we examine the effectiveness of adjunctive argon laser peripheral iridoplasty (ALPI) in patients who underwent NPT, to reduce post-operative IOP rise, secondary to IP obstructing outflow across the TDM. BACKGROUND: Nd:YAG laser goniopuncture consists of placing several laser shots on the undersurface of the trabeculodescemetic membrane. The result is a microperforation in this membrane, with flow of aqueous into the filtration bleb, which converts a non-perforating filtration procedure into a partial thickness filtration procedure. In this technique, several high power, multi-burst shots are applied, ab interno, by a Nd:YAG laser via a gonioprism, to the underside of the TDM, to facilitate aqueous outflow out from the anterior chamber. YGP can be effective when increased aqueous outflow is desired postoperatively. After this procedure, patent perforation in the TDM is usually observed gonioscopically, generally accompanied with reduction in IOP, and increase in bleb elevation (in height and in circumferential extent). Argon laser peripheral iridoplasty consists of placing several laser burns on the surface of the peripheral iris to contract the iris stroma, in a centripetal fashion, between the site of the burn and the anterior chamber angle. The result is iris stromal tissue contraction and compaction, movement of IP away from the angle and toward the pupil, which physically widens the angle and clears the synechial apposition of the peripheral iris against the TDM. In this technique, a series of low power, long duration, and large size burns is applied to the iris periphery to contract the iris stroma, to open the angle, and to clear IP causing synechial obstruction of the TDM after successful NPT. Used previously in acute angle closure glaucoma, ALPI may be effective in controlling IOP and clearing corneal edema when systemic and topical anti-glaucoma treatments fail to control high IOP, and when laser peripheral iridotomy (LPI) is not possible (e.g. in cases of severe corneal edema). Additionally, ALPI can be effective in permanently reopening the anterior chamber angle of iridectomized eyes with plateau iris syndrome; in this technique a full 360 degrees ring of spots is often applied, but a more limited area of treatment may also be effective. TECHNIQUE: When a post-operative elevation in IOP was detected in a patients eye which had undergone NPT, careful indentation gonioscopy was performed to examine the TDM at the surgical site. If the peripheral iris was flat, the anterior chamber angle was open, and the TDM did not appear obstructed by IP, YGP was performed. First, the eyes were pre-treated with aproclonodine 1% and pilocarpine 2% (if needed to allow visualization of the TDM). Next, a Nd:YAG laser was set on triple burst mode and shots were applied to the underside of the TDM at the NPT site, using a Goldman 3-mirror lens in the following manner: Energy - 3-5 mJ; Mode - Triple burst. The power and amount of spots were titrated in order to achieve partial or microperforation of TDM at the NPT surgical site, thus resulting in restoration of aqueous outflow into the filtration bleb. When a post-operative elevation in IOP was detected in a patients eye which had undergone NPT and or YGP, careful indentation gonioscopy was performed to examine the TDM at the surgical site. If irreducible synechial IP were detected, which obstructed filtration through the TDM at the NPT surgical site, ALPI was performed. First, the eyes were pre-treated with aproclonodine 1% and pilocarpine 2%. Next, an argon laser was set on blue-green mode and shots were applied to the IP adherent to cornea or to the TDM at the NPT site, using a Goldman 3-mirror lens in the following manner: Spot Size - 500 mM; Duration - 0.5 s; Power - 200 to 400 mW. The power and amount of burns were titrated in order to achieve partial or complete centripetal retraction of the IP from the TDM at the NPT surgical site, causing a clearance of the obstruction to the TDM, thus resulting in restoration of aqueous outflow into the filtration bleb.

[Comparison of human chorionic gonadotropin (Pregnyl 10 000 IU i.m.) versus GnRH agonist (triptorelin 0,2 mg s.c.) for final oocytes maturation in the same egg donors--clinical and embryological characteristics]. / Srovnání lidského choriového gonadotropinu (Pregnyl 10 000 IU i.m.) s GnRH agonistou (triptorelin 0.2 mg s.c.) pro maturaci oocytu u stejných dárkyn vajícek--klinické a embryologické charakteristiky.

OBJECTIVE: To compare clinical and embryological characteristics in donor cycles triggered for final oocytes maturation with Pregnyl 10 000 IU i.m. versus triptorelin 0.2 mg s.c. in the same patients in two sequential stimulation cycles. The aim of the study is to decrease the risk of the development of ovarian hyperstimulation syndrome (OHSS) at high response donors by the replacement of Pregnyl 10 000 IU i.m. vs. triptorelin 0.2 mg s.c. The administration of a single dose of gonadotropin-releasing hormone agonist (triptorelin 0.2 mg s.c.) induces release of LH from the pituitary gland similarly to a spontaneous LH surge. SUBJECT: Prospective cross-over trial. SETTING: Sanatorium Pronatal, Praha. SUBJECT AND METHOD: From August 2009 to July 2010 we analysed 24 stimulation cycles in 12 egg donors treated with GnRH antagonist protocol with recombinant FSH (follitropin beta). We identified patients with more than 15 follicles during examination by transvaginal ultrasound. When at least 3 leading follicles reached 17 mm in diameter we administrated Pregnyl 10 000 IU i.m. for final oocytes maturation and triptorelin 0.2 mg s.c in the subsequent treatment cycle. RESULTS: The primary outcome measure was number of oocytes, proportion mature oocytes and fertilized oocytes. The secondary outcome were duration of FSH stimulation, total dose of gonadotropins and mean daily dose of gonadotropins. Data was analysed by paired t-test. We retrieved 17.2 +/- 8.6 vs. 15.8 +/- 5.3 (ns) oocytes, 12.6 +/- 7.3 vs. 13.0 +/- 5.4 (ns) metaphase II oocytes, proportion of metaphase II oocytes (%) was 73 vs. 83 (ns), number of fertilized oocytes 11.5 +/- 6.7 vs. 11.7 +/- 4.5 (ns), fertilization rate (%) 91 vs. 90 (ns) in Pregnyl's vs. triptorelin's group, resp. Duration of FSH stimulation (days) 12.2 +/- 0.8 vs. 12.4 +/- 0.7 (ns), total dose of gonadotropins (IU) 1744 +/- 277 vs. 1740 +/- 276 (ns), mean daily dose of gonadotropins (IU) 238 +/- 43 vs. 221 +/- 36 (ns), were not statistically different in both groups. CONCLUSIONS: Number of mature oocytes and subsequent embryonic cleavage is comparable to standard hCG treatment. There are no differences in clinical and embryological characteristics in both groups. Only one patient with administration of Pregnyl 10 000 IU i.m. was treated for OHSS grade II by vaginal paracentesis. Administration of triptorelin 0.2 mg s.c. is a safe and effective approach to achieve mature oocytes in egg donation programme, where we do not take care of implantation, which has got some limitations based on several studies.

[Trauma to the triangular fibrocartilaginous complex (TFCC)]. / Traumatická poskození triangulárního fibrokartilaginózního komplexu (TFCC).

PURPOSE OF THE STUDY: The triangular fibrocartilaginous complex (TFCC) can be injured either due to trauma or by chronic strain. Based on these findings, Palmer devised a classification system distinguishing traumatic (I) and degenerative (II) TFCC lesions. Traumatic TFCC injury may be single or involved in a combined injury of the distal radius. The aim of this study was to evaluate the results of surgical treatment in patients with traumatic TFCC injury at six-month follow-up. MATERIAL AND METHODS: In the years 2000 to 2004, 23 patients with injury to the wrist were treated. The group comprised 16 men and 7 women at an average age of 34 years (range, 17 to 54 years). Nine patients were diagnosed with a distal radial fracture and 14 had no damage to the bony structures. Of these, seven showed clinical signs of TFCC injury and were indicated to acute arthroscopy of the wrist. The remaining seven, due to persisting complaints, were examined by arthroscopy within 3 months of injury. During the arthroscopic procedure, the Palmer type of injury was identified and arthroscopic treatment, open or closed, was carried out at the same stage. All patients were followed up for 6 months for pain relief and return to full physical activity. RESULTS: The arthroscopic examination revealed the following TFCC lesions: 11 central ruptures (I.A), 6 ulnar tears (I.B), 4 palmar (I.C) and 2 radial (I.D) avulsions. During arthroscopy, partial resection of the disc was performed in 13 cases (I.A, I.D), and reattachment of the disc to the styloid process of the ulna was carried out in six cases (I.B). In four of these patients the intervention was done at the stage of acute lesion within 4 weeks of injury, and in two within a longer period. Four patients with type I.C injuries underwent open disc reattachment from the palmar approach. Subjective evaluation showed that 65 % of the patients had no complaints, 26 % reported pain after excessive activity and 9 % had pain associated with daily activities. Nome of the patients reported rest pain. DISCUSSION: In our group, 91 % of the patients reported excellent and very good results at six months of follow-up. The two patients experiencing pain in daily activities (9 %) had type I.B. injury and were indicated for arthroscopy at a time longer than 6 weeks after injury. CONCLUSIONS: TFCC lesions are wrist injuries which, if diagnosed early and treated appropriately, show good healing. If the triangular fibrocartilage complex is damaged by a central tear, disc resection gives good results. However, if the distal radioulnar ligaments are torn, their reattachment is necessary in order to prevent instability of the distal radioulnar joint. Open surgical procedures interfere with integrity of the distal radioulnar joint ligaments, which may lead to joint instability and prolonged healing.

[Wrist arthroscopy]. / Artroskopie zápestí.

BACKGROUND: Pain in the wrist area is quite a frequent lesion. If it is not treated soon enough, it leads to development of degenerative changes which are curable with difficulty. Diagnosing is complicated because it is often difficult to diagnose them by means of radiological methods. Wrist arthroscopy is a method which enables to assess the problem and very often it also helps to solve it with an operation. METHODS AND RESULTS: Authors evaluated 93 wrist arthroscopies performed in their departments during 2004-2005. There were 28 arthroscopies performed due to an acute lesion and 65 arthroscopies due to chronic problems. There were 59 arthroscopic curative interventions performed during the operation and 64 open interventions were indicated on the basis of an arthroscopic examination. CONCLUSIONS: All wrist lesions where the lesion of connective tissues structures is suspected, accompanied with edema and hematoma, should be indicated for acute wrist arthroscopy, even though there is no finding on radiographs. Another group of patients indicated for the acute wrist arthroscopy are young patients with wrist fractures where there is a frequent ligament lesion associated, and patients with persisting ailments, even after radiologically confirmed healing of the fracture when intra-articular pathology can be expected. All patients with pain in the wrist area are indicated for arthroscopy, including those with not obvious cause of pain and with symptoms of arthrosis, because it is possible to evaluate further procedure of the therapy.

Storage of bovine isolated follicles: a new alternative way to improve the recovery rate of viable embryos from ovarian follicles of slaughtered cows.

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.

Development of a new Sindbis virus transducing system and its characterization in three Culicine mosquitoes and two Lepidopteran species.

Alphavirus transducing systems (ATSs) are alphavirus-based tools for expressing genes in insects. Here we describe an ATS (5'dsMRE16ic) based entirely on Sindbis MRE16 virus. GFP expression was used to characterize alimentary tract infections and dissemination in three Culicine and two Lepidopteran species. Following per os infection, 5'dsMRE16ic-EGFP efficiently infected Aedes aegypti and Culex tritaeniorhynchus, but not Culex pipiens pipiens. Ae. aegypti clearly showed accumulation of green fluorescent protein (GFP) in the posterior midgut and foregut/midgut junction within 2-3 days postinfection. Following parenteral infection of larvae, Bombyx mori had extensive GFP expression in larvae and adults, but Manduca sexta larvae were mostly resistant. 5'dsMRE16ic should be a valuable tool for gene expression in several important insect species that are otherwise difficult to manipulate genetically.

The allatostatin gene of the cricket Gryllus bimaculatus (Ensifera, Gryllidae).

The gene encoding allatostatins (AST) of the FGLamide family from the cricket Gryllus bimaculatus is expressed in the brain. The mRNA, which contains four polyadenylation signals, encodes a hormone precursor that is split into at least 14 putative hormones. Five of them have been previously found in the cricket, six to seven others, or their close homologues, are known from other insects. Hormone AST 2 contains an internal cleavage site and may exist in a shorter version 2b. The hormones AST 3 and 4 are identical. The cDNA sequence revealed that a single point mutation and a single deletion eliminated an additional hormone between AST 12 and 13. The deduced hormone precursor is very similar to that in cockroaches, but is different from a shorter precursor in locusts, indicating that the gene evolved very fast in the latter. Regions conserved between cockroaches and crickets include parts of the acidic spacers that separate clusters of hormones, suggesting that these spacers may have additional functions.

The conserved nuclear receptor Ftz-F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans.

BACKGROUND: Nuclear receptors are essential players in the development of all metazoans. The nematode Caenorhabditis elegans possesses more than 200 putative nuclear receptor genes, several times more than the number known in any other organism. Very few of these transcription factors are conserved with components of the steroid response pathways in vertebrates and arthropods. Ftz-F1, one of the evolutionarily oldest nuclear receptor types, is required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila. RESULTS: We employed two complementary approaches, direct mutagenesis and RNA interference, to explore the role of nhr-25, a C. elegans ortholog of Ftz-F1. Deletion mutants show that nhr-25 is essential for embryogenesis. RNA interference reveals additional requirements throughout the postembryonic life, namely in moulting and differentiation of the gonad and vulva. All these defects are consistent with the nhr-25 expression pattern, determined by in situ hybridization and GFP reporter activity. CONCLUSIONS: Our data link the C. elegans Ftz-F1 ortholog with a number of developmental processes. Significantly, its role in the periodical replacement of cuticle (moulting) appears to be evolutionarily shared with insects and thus supports the monophyletic origin of moulting.

Activation of a delayed-early gene encoding MHR3 by the ecdysone receptor heterodimer EcR-B1-USP-1 but not by EcR-B1-USP-2.

MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5' flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter fused to a chloramphenicol acetyltransferase (CAT) reporter by 2 micrograms of 20E per ml in Manduca GV1 cells was similar to that of endogenous MHR3, with detectable CAT by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) were expressed at high levels under the control of a constitutive promoter, CAT levels after a 3-h exposure to 20E increased two- to sixfold. In contrast, high expression of EcR-B1 and USP-2 caused little increase in CAT levels in response to 20E. Moreover, expression of USP-2 prevented activation by EcR-B1-USP-1. Deletion experiments showed that the upstream region, including the three most proximal putative EcREs, was responsible for most of the 20E activation, with the EcRE3 at -671 and the adjacent GGGTCA being most critical. The EcRE1 at -342 was necessary but not sufficient for the activational response but was the only one of the three putative EcREs to bind the EcR-B1-USP-1 complex in gel mobility shift assays and was responsible for the silencing action of EcR-B1-USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bound other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1-USP-2 heterodimer showed no binding to EcRE1, and the presence of excess USP-2 prevented the binding of EcR-B1-USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both formed heterodimeric complexes with EcR-B1 that bound ponasterone A with the same Kd (7 x 10(-10) M) and bound to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms.

Regulation of the transcription factor E75 by 20-hydroxyecdysone and juvenile hormone in the epidermis of the tobacco hornworm, Manduca sexta, during larval molting and metamorphosis.

The homolog of the ecdysteroid-induced transcription factor E75A in Drosophila melanogaster was cloned from the tobacco hornworm, Manduca sexta, and its developmental expression and hormonal regulation were analyzed. Both E75A and E75B mRNAs were found in the abdominal epidermis during both the larval and the pupal molts, with E75A appearing before E75B, coincident with the rise of ecdysteroid. Exposure of either fourth or fifth instar epidermis to 20E in vitro caused the rapid, transient induction of E75A RNA with a peak at 6 and 3 h, respectively, followed by maintenance at low levels until 24 h. Epidermis from fourth instar larvae with high endogenous juvenile hormone (JH) showed a 10-fold higher sensitivity to 20E (EC50 = 2 x 10(-8) M for fourth instar and 2 x 10(-7) M for fifth instar epidermis). The presence of the protein synthesis inhibitor anisomycin had no effect on the induction but prevented the decline, indicating that E75A RNA was directly induced by 20E, but its down-regulation depended on protein synthesis. Exposure of day 2 fifth instar epidermis to 20E in the presence of JH I, which prevents the 20E-induced pupal commitment, caused an increased accumulation of E75A RNA throughout the culture period although the temporal pattern was unaffected. These findings show for the first time that JH plays a role in 20E-induced early gene expression and suggest that the higher levels of E75A may be required for maintenance of larval commitment of this epidermis.

Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta.

cDNAs were isolated from Manduca sexta that encode two isoforms of an ultraspiracle (USP) homologue MsUSP-1 and MsUSP-2 with different N-terminal A/B regions. The MsUSP-1 cDNA predicts a protein with 97% and 45% amino acid identities in the DNA- and ligand-binding domains respectively to the Drosophila USP and 89% overall identity with Bombyx mori CF1 (an USP homologue). Northern blot hybridizations with probes specific to MsUSP-1 and MsUSP-2 showed transcripts of an approximately equal size (4.5 kb), but with diverse developmental profiles in Manduca epidermis during the two final larval instars and the onset of the adult moult. The MsUSP-1 mRNA was expressed during the intermoult periods, with higher levels around the time of the larval ecdyses and at the onset of wandering behaviour. In contrast, the MsUSP-2 mRNA was up-regulated at times of high ecdysteroid titre during the larval moults, when the MsUSP-1 mRNA disappeared. Together, these conversely regulated isoform mRNAs contribute to the constitutive expression profile of total MsUSP mRNA.

Developmental expression of Ultraspiracle proteins in the epidermis of the tobacco hornworm, Manduca sexta, during larval life and the onset of metamorphosis.

During the final two larval instars, a changing pattern of three Ultraspiracle (Usp) proteins (50.5, 52.5, and 57 kDa) was detected in immunoblots of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, by a monoclonal antibody against Drosophila Usp that was shown to detect MsUsp. The 57- and 52.5-kDa bands were present during the intermolt periods and the 50.5- and 52.5-kDa bands during the molting phases. The antibody detected a nuclear antigen present in epidermis, muscle, fat body, and the central nervous system from the time of hatching. In the epidermis Usp was present in all cell nuclei but was especially prominent in the tormogen and trichogen cells immediately after ecdysis in both the penultimate and final instars. This latter immunoreactivity disappeared within 12 h whereas the remainder of the epidermis retained high levels throughout the feeding period. During the molt immunostaining reappeared in the hair cell nuclei. During the wandering stage at the onset of metamorphosis and just before pupal ecdysis, immunoblots showed high levels of Usp, but nuclei showed little or no staining. This discrepancy is likely due to the loss of one Usp isoform from the nucleus and its dispersal in the cytoplasm in preparation for the appearance of the second isoform.

Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta.

Ecdysteroids acting through multiple isoforms of the ecdysone receptor (EcR) initiate molting and metamorphosis of insects. Two isoforms of EcR, A (this paper) and B1 (Fujiwara et al., Insect Biochem. Mol. Biol. 25, 845-856, 1995), were isolated from the tobacco hornworm, Manduca sexta, and shown to be similar to the corresponding Drosophila EcR isoforms. The developmental profiles of both EcR-A and EcR-B1 (determined by both analysis of isoform-specific mRNAs and use of monoclonal antibodies that detect either EcR-B1 or all forms), however, were different in Manduca epidermis, which produces sequentially the larval, the pupal, and the adult cuticles. EcR-B1 predominated through the larval, pupal, and early developing adult stages with an upregulation early in each molt. By contrast, EcR-A was present only at the onset of new cuticle synthesis during the larval molt, but in the pupal and adult molts was upregulated slightly later than EcR-B1 during the commitment period and was present during the predifferentiative phase. Both isoforms appeared in the larval wing discs after pupal commitment and persisted through pupal differentiation. The mRNAs for both isoforms were directly induced in larval epidermis in vitro by 20-hydroxyecdysone, but EcR-B1 mRNA accumulated more rapidly, peaking at 3 hr. In the presence of a protein synthesis inhibitor, the accumulation of EcR-B1 mRNA was slower and its subsequent decline was prevented, but the accumulation of EcR-A mRNA was unaffected. Thus, in this polymorphic epidermis both isoforms appear in every molt, with EcR-B1 present during the commitment and predifferentiative phases and then at the onset of cuticle synthesis EcR-A prevails. Additionally, EcR-A is apparently associated with the switching and predifferentiative events necessary for a new synthetic program.

Expression of ecdysteroid-regulated transcripts in the silk gland of the wax moth, Galleria mellonella.

Expression of six different transcripts encoding members of the nuclear receptor superfamily was studied in whole silk glands of the wax moth, Galleria mellonella, during development and in response to 20-hydroxyecdysone (20E) and juvenile hormone (JH II) in vitro. Nucleic acid probes specific to Galleria homologs of the Drosophila E75 early gene and the delayed early gene DHR3 were used to follow mRNAs encoding isoforms A and B of the E75 protein and GHR3 protein, respectively. Among these mRNAs, E75A mRNA appeared first during the larval molt and was most rapidly induced by 20E, while E75B and GHR3 mRNAs appeared with a 2 h delay. Unlike E75B mRNA, GHR3 mRNA required protein synthesis for its full induction. The 20E-induced levels of E75A mRNA were increased one and a half-fold within 1 h in the presence of 10-100 ng/ml JH II in cultured silk glands. Antisense cRNA probes for the Manduca sexta ecdysone receptor isoforms EcR-A and EcR-B1 detected transcripts that were directly induced by 20E in cultured silk glands. The levels of the putative EcR-A mRNA were high relative to those of EcR-B1 mRNA in silk glands at molting and metamorphosis. An antisense cRNA probe for the Manduca Ultraspiracle homolog detected a 4 kb mRNA that was most abundant around the time of larval ecdysis but was not induced by 20E in vitro.

Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings.

Using the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA clone, we isolated three genomic clones for EcR from the tobacco hornworm, Manduca sexta. Subsequent isolation and sequencing of several cDNAs yielded a homolog of the B1 isoform with 50, 95 and 70% amino acid identities with DmEcR in the N-terminal A/B, the DNA binding and the ligand binding domains respectively. Unlike Drosophila, an intron occurs between the exons encoding the two zinc fingers of Manduca EcR (MsEcR). A 6.0 kb mRNA encoding MsEcR was found in both larval wing discs and prothoracic glands and in pupal wings. During the final larval instar, the mRNA was maximal in the wing discs at one day after wandering (W1), whereas in the prothoracic gland EcR mRNA increased rapidly to high levels on day 2 and remained high thereafter. During the onset of adult development, two peaks of EcR mRNA were observed in wings from day 3 to 5 and on day 8 after pupal ecdysis. These two peaks correlated with the time of increasing titers of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The EcR mRNA peaks always preceded the large ecdysteroid peak, suggesting that the transcription of the EcR gene is induced by a low concentration of ecdysteroid in vivo.

Isolation and developmental expression of the ecdysteroid-induced GHR3 gene of the wax moth Galleria mellonella.

Three degenerate primers were designed to match the most conserved regions within the DNA-binding domains of several selected members of the steroid hormone receptor family. Use of these primers in the polymerase chain reaction with cDNA from Galleria mellonella prepupae detected a 177 bp fragment that had 87% identity to the Manduca sexta gene MHR3 and 75% to the Drosophila melanogaster DHR3 gene, and therefore was named "GHR3". Screening of a Galleria penultimate instar cDNA library with this fragment yielded a cDNA clone that contained a 557 codon open reading frame, predicting a 62.3 kDa protein. The deduced amino acid sequence of GHR3 showed 92% overall identity with the MHR3 protein and 97 and 70% identity with DHR3 in the putative DNA- and ligand-binding domains, respectively. Hybridization of whole body RNA revealed high GHR3 mRNA levels during both the larval and pupal molts, coincident with the molt-inducing ecdysteroid pulses, and low or undetectable levels during the first half of the last instar. During the larval-pupal transformation, no GHR3 mRNA was found at the beginning of the stemmatal pigment retraction at the onset of the ecdysteroid rise; maximal levels were observed 4 h later, coincident with the peak ecdysteroid titer (over 2.3 micrograms 20E equivalents/ml hemolymph). Two mRNAs (4.6 and 3.6 kb) were detected when the ecdysteroid titer was high. Injection of 2 micrograms/gm 20E into isolated final instar larval abdomens induced the appearance of the 4.6 kb mRNA within 1.5 h; the mRNA level then reached maximum by 3 h and declined by 6 h. No 3.6 kb mRNA was detectable during that time. A 10-fold lower 20E dose caused only trace induction by 3 h.

Isolation, characterization and developmental expression of the ecdysteroid-induced E75 gene of the wax moth Galleria mellonella.

Using the cDNA for the Drosophila ecdysteroid-induced member of the steroid-hormone-receptor superfamily, E75A, we isolated a genomic clone from Galleria mellonella that revealed 77% similarity with the region of E75A cDNA encoding the C-terminal zinc-finger motif. A Galleria cDNA clone was isolated that encoded a complete DNA-binding domain composed of two zinc fingers and designated GmE75A. Its deduced amino acid sequence showed 100% and 85% identities within the DNA-binding and ligand-binding domains of Drosophila E75A, respectively. The Galleria genomic clone did not encode the N-terminal zinc finger, but included a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicated that the Galleria gene E75 encoded at least two isoforms, GmE75A and GmE75B, which differed in their N-termini. Probes specific for GmE75A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75A and B mRNA levels correlated closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appeared in high amounts within 4 h of the ecdysteroid rise, then declined concurrently with the hormone titer decline. At the time of pupal ecdysis, there was another peak of GmE75A expression but not GmE75B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75A mRNA was induced by 20-hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment.