Search

Background: Photosynthesis is one of the most important biological reactions and forms the basis of crop productivity and yield on which a growing global population relies. However, to develop improved plant cultivars that are capable of increased productivity, methods that can accurately and quickly quantify photosynthetic efficiency in large numbers of genotypes under field conditions are needed. Chlorophyll fluorescence imaging is a rapid, non-destructive measurement that can provide insight into the efficiency of the light-dependent reactions of photosynthesis., Results: To test and validate a field-deployed fluorescence imaging system on the TERRA-REF field scanalyzer, leaves of potted sorghum plants were treated with a photosystem II inhibitor, DCMU, to reduce photochemical efficiency (F V /F M ). The ability of the fluorescence imaging system to detect changes in fluorescence was determined by comparing the image-derived values with a handheld fluorometer. This study demonstrated that the imaging system was able to accurately measure photochemical efficiency (F V /F M ) and was highly correlated ( r  = 0.92) with the handheld fluorometer values. Additionally, the fluorescence imaging system was able to track the decrease in photochemical efficiency due to treatment of DCMU over a 7 day period., Conclusions: The system's ability to capture the temporal dynamics of the plants' response to this induced stress, which has comparable dynamics to abiotic and biotic stressors found in field environments, indicates the system is operating correctly. With the validation of the fluorescence imaging system, physiological and genetic studies can be undertaken that leverage the fluorescence imaging capabilities and throughput of the field scanalyzer., Competing Interests: Competing interestsThe authors declare no conflicts of interest. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity employer., (© The Author(s) 2020.)

Background: Guayule (Parthenium argentatum Gray) is a drought tolerant, rubber producing perennial shrub native to northern Mexico and the US Southwest. Hevea brasiliensis, currently the world's only source of natural rubber, is grown as a monoculture, leaving it vulnerable to both biotic and abiotic stressors. Isolation of rubber from guayule occurs by mechanical harvesting of the entire plant. It has been reported that environmental conditions leading up to harvest have a profound impact on rubber yield. The link between rubber biosynthesis and drought, a common environmental condition in guayule's native habitat, is currently unclear., Results: We took a transcriptomic and comparative genomic approach to determine how drought impacts rubber biosynthesis in guayule. We compared transcriptional profiles of stem tissue, the location of guayule rubber biosynthesis, collected from field-grown plants subjected to water-deficit (drought) and well-watered (control) conditions. Plants subjected to the imposed drought conditions displayed an increase in production of transcripts associated with defense responses and water homeostasis, and a decrease in transcripts associated with rubber biosynthesis. An evolutionary and comparative analysis of stress-response transcripts suggests that more anciently duplicated transcripts shared among the Asteraceae, rather than recently derived duplicates, are contributing to the drought response observed in guayule. In addition, we identified several deeply conserved long non-coding RNAs (lncRNAs) containing microRNA binding motifs. One lncRNA in particular, with origins at the base of Asteraceae, may be regulating the vegetative to reproductive transition observed in water-stressed guayule by acting as a miRNA sponge for miR166., Conclusions: These data represent the first genomic analyses of how guayule responds to drought like conditions in agricultural production settings. We identified an inverse relationship between stress-responsive transcripts and those associated with precursor pathways to rubber biosynthesis suggesting a physiological trade-off between maintaining homeostasis and plant productivity. We also identify a number of regulators of abiotic responses, including transcription factors and lncRNAs, that are strong candidates for future projects aimed at modulating rubber biosynthesis under water-limiting conditions common to guayules' native production environment.

Systemic Treatment of Eating Disorders Abstract. Eating disorders (EDs) are deleterious illnesses that are associated with significant psychiatric and medical morbidity and mortality, considerable distress and impairment, marked caregiver burden, and high treatment costs. Because EDs commonly onset in adolescence and young adulthood, and with consistent evidence that early intervention results in the most promising treatment outcomes, an increasing amount of research has been devoted to the treatment of adolescent EDs. Although still less researched in adult presentations of EDs, the historical record of research on adolescent ED treatment over the last half-century principally supports family therapy. Current published clinical guidelines recommend an ED-specific family therapy as the first-line treatment of adolescents with anorexia nervosa (AN) and as a recommended treatment of adolescents with bulimia nervosa (BN). The number of treatment trials for adolescent AN has slowly grown over the last few decades and, more recently, family interventions include protocols extending to new populations and diagnoses, including BN. This narrative review summarizes existing family-based approaches to the treatment of adolescent EDs, integrating recent research findings. This article also includes discussion of methods, both current and proposed, that expand and adapt current family-based approaches in efforts to improve the breadth and scope of ED treatment in adolescence and young adulthood.

Adolescents seeking professional help with their gender identity development often present with psychological difficulties. Existing literature on psychological functioning of gender diverse young people is limited and mostly bound to national chart reviews. This study examined the prevalence of psychological functioning and peer relationship problems in adolescents across four European specialist gender services (The Netherlands, Belgium, the UK, and Switzerland), using the Child Behavioural Checklist (CBCL) and the Youth Self-Report (YSR). Differences in psychological functioning and peer relationships were found in gender diverse adolescents across Europe. Overall, emotional and behavioural problems and peer relationship problems were most prevalent in adolescents from the UK, followed by Switzerland and Belgium. The least behavioural and emotional problems and peer relationship problems were reported by adolescents from The Netherlands. Across the four clinics, a similar pattern of gender differences was found. Birth-assigned girls showed more behavioural problems and externalising problems in the clinical range, as reported by their parents. According to self-report, internalising problems in the clinical range were more prevalent in adolescent birth-assigned boys. More research is needed to gain a better understanding of the difference in clinical presentations in gender diverse adolescents and to investigate what contextual factors that may contribute to this.

To mitigate the effects of heat and drought stress, a better understanding of the genetic control of physiological responses to these environmental conditions is needed. To this end, we evaluated an upland cotton ( Gossypium hirsutum L.) mapping population under water-limited and well-watered conditions in a hot, arid environment. The elemental concentrations (ionome) of seed samples from the population were profiled in addition to those of soil samples taken from throughout the field site to better model environmental variation. The elements profiled in seeds exhibited moderate to high heritabilities, as well as strong phenotypic and genotypic correlations between elements that were not altered by the imposed irrigation regimes. Quantitative trait loci (QTL) mapping results from a Bayesian classification method identified multiple genomic regions where QTL for individual elements colocalized, suggesting that genetic control of the ionome is highly interrelated. To more fully explore this genetic architecture, multivariate QTL mapping was implemented among groups of biochemically related elements. This analysis revealed both additional and pleiotropic QTL responsible for coordinated control of phenotypic variation for elemental accumulation. Machine learning algorithms that utilized only ionomic data predicted the irrigation regime under which genotypes were evaluated with very high accuracy. Taken together, these results demonstrate the extent to which the seed ionome is genetically interrelated and predictive of plant physiological responses to adverse environmental conditions., (Copyright © 2018 Pauli et al.)

Sodium (Na + ) accumulation in the cytosol will result in ion homeostasis imbalance and toxicity of transpiring leaves. Studies of salinity tolerance in the diploid wheat ancestor Triticum monococcum showed that HKT1;5 -like gene was a major gene in the QTL for salt tolerance, named Nax2 . In the present study, we were interested in investigating the molecular mechanisms underpinning the role of the HKT1;5 gene in salt tolerance in barley ( Hordeum vulgare ). A USDA mini-core collection of 2,671 barley lines, part of a field trial was screened for salinity tolerance, and a Genome Wide Association Study (GWAS) was performed. Our results showed important SNPs that are correlated with salt tolerance that mapped to a region where HKT1;5 ion transporter located on chromosome four. Furthermore, sodium (Na + ) and potassium (K + ) content analysis revealed that tolerant lines accumulate more sodium in roots and leaf sheaths, than in the sensitive ones. In contrast, sodium concentration was reduced in leaf blades of the tolerant lines under salt stress. In the absence of NaCl, the concentration of Na + and K + were the same in the roots, leaf sheaths and leaf blades between the tolerant and the sensitive lines. In order to study the molecular mechanism behind that, alleles of the HKT1;5 gene from five tolerant and five sensitive barley lines were cloned and sequenced. Sequence analysis did not show the presence of any polymorphism that distinguishes between the tolerant and sensitive alleles. Our real-time RT-PCR experiments, showed that the expression of HKT1;5 gene in roots of the tolerant line was significantly induced after challenging the plants with salt stress. In contrast, in leaf sheaths the expression was decreased after salt treatment. In sensitive lines, there was no difference in the expression of HKT1;5 gene in leaf sheath under control and saline conditions, while a slight increase in the expression was observed in roots after salt treatment. These results provide stronger evidence that HKT1;5 gene in barley play a key role in withdrawing Na + from the xylem and therefore reducing its transport to leaves. Given all that, these data support the hypothesis that HKT1;5 gene is responsible for Na + unloading to the xylem and controlling its distribution in the shoots, which provide new insight into the understanding of this QTL for salinity tolerance in barley.

Many systems for field-based, high-throughput phenotyping (FB-HTP) quantify and characterize the reflected radiation from the crop canopy to derive phenotypes, as well as infer plant function and health status. However, given the technology's nascent status, it remains unknown how biophysical and physiological properties of the plant canopy impact downstream interpretation and application of canopy reflectance data. In that light, we assessed relationships between leaf thickness and several canopy-associated traits, including normalized difference vegetation index (NDVI), which was collected via active reflectance sensors carried on a mobile FB-HTP system, carbon isotope discrimination (CID), and chlorophyll content. To investigate the relationships among traits, two distinct cotton populations, an upland ( Gossypium hirsutum L.) recombinant inbred line (RIL) population of 95 lines and a Pima ( G. barbadense L.) population composed of 25 diverse cultivars, were evaluated under contrasting irrigation regimes, water-limited (WL) and well-watered (WW) conditions, across 3 years. We detected four quantitative trait loci (QTL) and significant variation in both populations for leaf thickness among genotypes as well as high estimates of broad-sense heritability (on average, above 0.7 for both populations), indicating a strong genetic basis for leaf thickness. Strong phenotypic correlations (maximum r = -0.73) were observed between leaf thickness and NDVI in the Pima population, but not the RIL population. Additionally, estimated genotypic correlations within the RIL population for leaf thickness with CID, chlorophyll content, and nitrogen discrimination ([Formula: see text] = -0.32, 0.48, and 0.40, respectively) were all significant under WW but not WL conditions. Economically important fiber quality traits did not exhibit significant phenotypic or genotypic correlations with canopy traits. Overall, our results support considering variation in leaf thickness as a potential contributing factor to variation in NDVI or other canopy traits measured via proximal sensing, and as a trait that impacts fundamental physiological responses of plants.

Background  High-pressure injection injuries are devastating injuries that often lead to an amputation of the primarily affected finger. The available literature mainly focuses on amputation rates, while there is a lack of knowledge regarding functional results. Patients and Methods  Between 1998 and 2016, 47 cases of high-pressure injection injuries of the hand were registered in our department. Out of these, data of 26 patients, all men with an average age of 42 (18-62) years, were included in a retrospective follow-up examination after 4 (1-12) years. We recorded DASH scores, pain intensity on visual analogue scales (VAS, 1-10), fingertip-to-palm distance, active range of wrist motion, grip strength, and two-point-discrimination ability (2PD). The statistical analysis was descriptive and was performed by analysis of correlation. Results  The average DASH score of these 26 patients was 45 (30-93), pain intensity was 2.5 (0-9) on the VAS, fingertip-to-palm distance was 1 (0-4) cm. Active range of wrist motion was 99 (77-122) % of the contralateral side. Average grip strength was reduced to 67 (0-164) % of the contralateral side. Average 2PD was 9 (2-25) mm. Nineteen out of 26 patients returned to work. An amputation was necessary in 7 out of 26 cases. These patients had more pain and less grip strength. High latency from injury to operative treatment impaired functional outcome, which was statistically significant for DASH (p < 0.05). A trend to poorer outcomes was noted in cases of a more distal lesion and injection of toxic substances, whereas high injection pressure had no negative influence. Conclusion  High-pressure injection injuries have serious consequences regarding hand function and frequently cause chronic pain. This affects patients with finger preservation as well as patients requiring an amputation. A further evaluation of prognostic factors requires a metaanalysis of functional parameters. The DASH score seems to be an expedient assessment tool., Competing Interests: Interessenkonflikt: Die Autoren geben an, dass kein Interessenkonflikt besteht., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Transcriptional elongation is a generic function, but is also regulated to allow rapid transcription responses. Following relatively long initiation and promoter clearance, RNA polymerase II can pause and then rapidly elongate following recruitment of positive elongation factors. Multiple elongation complexes exist, but the role of specific components in adult Drosophila is underexplored., Results: We conducted RNA-seq experiments to analyze the effect of RNAi knockdown of Suppressor of Triplolethal and lilliputian. We similarly analyzed the effect of expressing a dominant negative Cyclin-dependent kinase 9 allele. We observed that almost half of the genes expressed in adults showed reduced expression, supporting a broad role for the three tested genes in steady-state transcript abundance. Expression profiles following lilliputian and Suppressor of Triplolethal RNAi were nearly identical raising the possibility that they are obligatory co-factors. Genes showing reduced expression due to these RNAi treatments were short and enriched for genes encoding metabolic or enzymatic functions. The dominant-negative Cyclin-dependent kinase 9 profiles showed both overlapping and specific differential expression, suggesting involvement in multiple complexes. We also observed hundreds of genes with sex-biased differential expression following treatment., Conclusion: Transcriptional profiles suggest that Lilliputian and Suppressor of Triplolethal are obligatory cofactors in the adult and that they can also function with Cyclin-dependent kinase 9 at a subset of loci. Our results suggest that transcriptional elongation control is especially important for rapidly expressed genes to support digestion and metabolism, many of which have sex-biased function.

Background: Understanding motivation to change is a key issue in both the assessment and the treatment of eating disorders. Therefore, sound instruments assessing this construct are of great help to clinicians. Accordingly, the present study analysed the psychometric properties of the Anorexia Nervosa Stages of Change Questionnaire (ANSOCQ), including its relation to coping style and self-esteem., Methods: N  = 92 adolescents referred to an eating disorders outpatient clinic meeting criteria for anorexia nervosa gave written informed consent to participate in this study and completed the ANSOCQ, the Eating Disorder Inventory, the Eating Attitudes Test, the Body Image Questionnaire, two questionnaires measuring Self-Related Cognitions and the Coping Across Situations Questionnaire. After a treatment period of nine months, clinical anorexia nervosa diagnosis and the body mass index were re-assessed. In addition to exploratory factor analysis, correlational analysis was used to test for the convergent validity of the ANSOCQ and logistic regression analysis was used to test its predictive validity., Results: The ANSOCQ had good psychometric properties. Factor analysis yielded two meaningful factors labelled as 'weight gain and control' and 'attitudes and feelings'. Internal consistencies of the two factors amounted to Cronbach's alpha = .87 and .76, respectively. Significant correlations with other scales measuring eating disorder psychopathology were indicative of meaningful construct validity. Higher motivation to change was related to higher self-esteem and a more active coping style. Higher (positive) ANSOCQ total scores predicted remission of anorexia nervosa after nine months of treatment. A higher score on 'attitudes and feelings' was a protective factor against drop-out from intervention., Conclusions: The ANSOCQ is a clinically useful instrument for measuring motivation to change in adolescents with AN. Two factorial dimensions explain most of the variation. Self-esteem and coping style are relevant additional constructs for the understanding of the motivation to change in anorexia nervosa., Trial Registration: NCT02828956. Retrospectively registered July 2016.

The application of high-throughput plant phenotyping (HTPP) to continuously study plant populations under relevant growing conditions creates the possibility to more efficiently dissect the genetic basis of dynamic adaptive traits. Toward this end, we employed a field-based HTPP system that deployed sets of sensors to simultaneously measure canopy temperature, reflectance, and height on a cotton (Gossypium hirsutum L.) recombinant inbred line mapping population. The evaluation trials were conducted under well-watered and water-limited conditions in a replicated field experiment at a hot, arid location in central Arizona, with trait measurements taken at different times on multiple days across 2010-2012. Canopy temperature, normalized difference vegetation index (NDVI), height, and leaf area index (LAI) displayed moderate-to-high broad-sense heritabilities, as well as varied interactions among genotypes with water regime and time of day. Distinct temporal patterns of quantitative trait loci (QTL) expression were mostly observed for canopy temperature and NDVI, and varied across plant developmental stages. In addition, the strength of correlation between HTPP canopy traits and agronomic traits, such as lint yield, displayed a time-dependent relationship. We also found that the genomic position of some QTL controlling HTPP canopy traits were shared with those of QTL identified for agronomic and physiological traits. This work demonstrates the novel use of a field-based HTPP system to study the genetic basis of stress-adaptive traits in cotton, and these results have the potential to facilitate the development of stress-resilient cotton cultivars., (Copyright © 2016 Pauli et al.)

Adolescence is associated with a dramatic surge in suicide rate. While worldwide age-adjusted suicides rate per 100’000 in under 14-year olds are relatively low around 0.6, a dramatic increase to 7.4 can be observed in 15 – 19 year olds with suicide being one of the three leading causes of mortality in most countries in this age group. From 2000 to 2013 the Swiss annual average of completed suicides in 15 – 19 year olds was 33.1 (minimum 24; maximum 47). This corresponds to an average age-specific death rate of 7.6, which is slightly above the global mean. Suicide prevention and treatment of suicidality in adolescents is a complex underpinning. Subjectively experienced feelings of insufficiency, hopelessness and the feeling of worthlessness combined with concrete suicidal ideations and the capacity to act out are important indicators for assessing suicidal risk. Suicide attempts as the strongest risk factor for suicide should always be taken seriously, even if they occur in the context of situational crisis. Narcissistic personality traits or increased impulsivity are further indicators of risk. The early detection and effective treatment of mental disorders such as depression, anxiety disorders, bipolar-affective disorders or psychotic disorders provide a great opportunity to reduce the frequency of suicide attempts and suicide. Particular caution is required when co-morbid addictions are present. The maintenance or establishment of sustainable relationships with significant others is considered a key protecting factor. Increasing withdrawal from significant others is an important warning sign and needs urgent involvement of experts. In acute suicidality, the admission to a psychiatric hospital must be considered, if necessary against the will of affected adolescent and their relatives. Personality-related recurrent suicidal behavior requires specific treatment approaches taking into account the prevention of iatrogenic worsening of suicidal patterns.

A recently introduced new SPS resin, possessing a 2-(ortho-nitrophenyl)ethanal linker, was used for the regioselective on-resin synthesis of N-mono-hydroxylated and N-mono-methylated polyamine spider toxins of Agelenopsis aperta and Larinioides folium. The polyamine backbones of the target compounds were efficiently constructed from the center by reductive amination of the aldehyde linker, followed by stepwise alkylation and acylation on solid support. Depending on the cleavage conditions, employing either oxidation/Cope elimination or methylation/Hofmann elimination, regioselectively the respective N-hydroxyl or N-methyl products were obtained. Employing this methodology, a number of acylpolyamine spider toxins were synthesized and identified as venom components by UHPLC and ESI-MS/MS.

The GAGA factor (GAF), encoded by the Trithorax like gene (Trl) is a multifunctional protein involved in gene activation, Polycomb-dependent repression, chromatin remodeling and is a component of chromatin domain boundaries. Although first isolated as transcriptional activator of the Drosophila homeotic gene Ultrabithorax (Ubx), the molecular basis of this GAF activity is unknown. Here we show that dmTAF3 (also known as BIP2 and dTAF(II)155), a component of TFIID, interacts directly with GAF. We generated mutations in dmTAF3 and show that, in Trl mutant background, they affect transcription of Ubx leading to enhancement of Ubx phenotype. These results reveal that the gene activation pathway involving GAF is through its direct interaction with dmTAF3.

Background: Umbilical cord blood (UCB) can be used as hematopoietic stem cell source for transplantation. The success of a transplantation is highly correlated with the number of total nucleated cells (TNCs) and CD34+ cells in the UCB. Certain obstetric factors increase the yield of stem cells in the UCB. It is necessary to evaluate optimal conditions in labor to decrease the rate of sample rejection due to low cell count. No data exist regarding the difference between primary and secondary Cesarean sections in terms of efficacy of stem cell harvesting., Study Design and Methods: Seventy-nine consecutive UCB units from women who had a Cesarean section between 1997 and 2003 were included. The number of TNCs, CD34+ cells, colony-forming units (CFUs), white blood cells (WBCs), nucleated red blood cells (NRBCs), and the total collection volume were compared between cases with primary and secondary Cesarean section., Results: UCB obtained after a Cesarean section due to fetal distress has significantly higher numbers of TNCs, CD34+ cells, NRBCs, and WBCs compared to elective Cesarean section. Of the cases with secondary Cesarean section due to fetal distress, 67 percent resulted in UCB units with sufficient TNC numbers (> or =80 x 10(7) TNCs) compared to 42 percent of the cases with primary Cesarean section., Conclusion: Fetal distress increases the number of hematopoietic stem cells mobilized into UCB. Particular effort should be made to collect UCB from newborns who experienced fetal distress.

Applying basic potentiometric and photometric assays, we evaluated the fully automated random access chemistry analyzer Architect c8000, a new member of the Abbott Architect system family, with respect to both its analytical and operational performance and compared it to an established high-throughput chemistry platform, the Abbott Aeroset. Our results demonstrate that intra- and inter-assay imprecision, inaccuracy, lower limit of detection and linear range of the c8000 generally meet actual requirements of laboratory diagnosis; there were only rare exceptions, e.g. assays for plasma lipase or urine uric acid which apparently need to be improved by additional rinsing of reagent pipettors. Even with plasma exhibiting CK activities as high as 40.000 U/l, sample carryover by the c8000 could not be detected. Comparison of methods run on the c8000 and the Aeroset revealed correlation coefficients of 0.98-1.00; if identical chemistries were applied on both analyzers, slopes of regression lines approached unity. With typical laboratory workloads including 10-20% STAT samples and up to 10% samples with high analyte concentrations demanding dilutional reruns, steady-state throughput numbers of 700 to 800 tests per hour were obtained with the c8000. The system generally responded to STAT orders within 2 minutes yielding analytical STAT order completion times of 5 to 15 minutes depending on the type and number of assays requested per sample. Due to its extended test and sample processing capabilities and highly comfortable software, the c8000 may meet the varying needs of clinical laboratories rather well.

The Su(var)3-7 gene, a haplo-suppressor and triplo-enhancer of position-effect variegation (PEV), encodes a zinc finger heterochromatin-associated protein. To understand the role of this protein in heterochromatin and genomic silencing, mutations were generated by homologous recombination. The donor fragment contained a yellow(+) gene and 7.6 kb of the Su(var)3-7 gene inserted between two FRTs. The Su(var)3-7 sequence contained three stop codons flanking an I-SceI cut site located in the 5' half of the gene. Using two different screening approaches, we obtained an allelic series composed of three mutant alleles. The three mutations are dominant suppressors of PEV. One behaves as a null mutation and results in a maternal-effect recessive lethal phenotype that can be rescued by a zygotic paternal wild-type gene. A P transposon zygotically expressing a Su(var)3-7 full-length cDNA also rescues the mutant phenotype. One hypomorphic allele is viable and the pleiotropic phenotype showed by adult flies indicates that rapidly and late dividing cells seem the most affected by reduced amounts of Su(var)3-7 protein. All three mutants were characterized at the molecular level. Each expresses a portion of the Su(var)3-7 protein that is unable to enter the nucleus and bind chromatin.

We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

The ovo+ and ovarian tumor+ genes function in the germline sex determination pathway in Drosophila, but the hierarchical relationship between them is unknown. We found that increased ovo+ copy number resulted in increased ovarian tumor expression in the female germline and increased ovo expression in the male germline. The ovo locus encodes C2H2 zinc-finger proteins. Bacterially expressed OVO zinc-finger domain bound to multiple sites at or near the ovo and ovarian tumor promoters strongly suggesting that OVO is directly autoregulatory and that ovarian tumor is a direct downstream target of ovo in the germline sex determination hierarchy. Both positive and negative regulation by OVO proteins appears likely, depending on promoter context and on the sex of the fly. Our observation that two strong OVO-binding sites are at the initiator of the TATA-less ovo-B and ovarian tumor promoters raises the possibility that OVO proteins influence the nucleation of transcriptional pre-initiation complexes.

We report the cloning of the noisette gene (noi), which encodes the Drosophila melanogaster ortholog of a U2 snRNP-associated splicing factor, SF3a60 (SAP61) in humans and PRP9p in Saccharomyces cerevisiae. Antibodies raised against human SF3a60 recognized NOI in flies, showing a nuclear localization in all the stages examined, including the embryo, the dividing cells of imaginal discs, and the larval polyploid nuclei. NOI is expressed in somatic and germinal cells of both male and female gonads. By mobilization of P transposons, we have generated a large number of noi mutations. Complete loss of function resulted in lethality at the end of embryogenesis, without obvious morphological defects. Hypomorphic alleles revealed multiple roles of noi for the survival and differentiation of male germ cells, the differentiation of female germ cells, and the development of several adult structures.

Mutations in ovo result in several different phenotypes, which we show are due to the regulation of distinct developmental pathways. Two X (female) germ cells require ovo+ activity for viability, but 1X (male) germ cells do not. In our study, we observed suppression of the ovo germline-lethality phenotype in loss-of-function maleless (mle) females indicating that ovo+ and mle+ have opposing effects in female germ cells; or that they are hierarchically related. Gain-of-function Sex-lethal (Sxl) alleles and male specific lethal-2 alleles did not suppress the ovo germline death phenotype. Many of the surviving germ cells in females mutant for both ovo and mle showed ovarian tumors. In contrast to the germline viability phenotype, we did observe suppression of the tumor phenotype in females heterozygous for gain-of-function alleles of Sxl. Further, females mutant for some hypomorphic ovo alleles were rendered fertile by Sxl gain-of-function alleles. Thus, ovo+ is required for at least two distinct functions, one involving mle+, and one mediated by Sxl+ gene products. The existence of ovo+ functions independent of mle+ and Sxl+ is likely.

We identified a new gene, stand still (stil), required in the female germline for proper survival, sex determination and differentiation. Three strong loss-of-function alleles were isolated. The strongest phenotype exhibited by ovaries dissected from adult females is the complete absence of germ cells. In other ovaries, the few surviving germ cells frequently show a morphology typical of primary spermatocytes. stil is not required either for fly viability or for male germline development. The gene was cloned and found to encode a novel protein. stil is strongly expressed in the female germ cells. Using P[stil+] transgenes, we show that stil and a closely localized gene are involved in the modification of the ovarian phenotypes of the dominant alleles of ovo caused by heterozygosity of region 49 A-D. The similarity of the mutant phenotypes of stil to that of otu and ovo suggests that the three genes function in a common or in parallel pathways necessary in the female germline for its survival, sex determination and differentiation.

Both position-effect variegation (PEV) in Drosophila and telomeric position-effect in yeast (TPE) result from the mosaic inactivation of genes relocated next to a block of centromeric heterochromatin or next to telomeres. In many aspects, these phenomena are analogous to other epigenetic silencing mechanisms, such as the control of homeotic gene clusters, X-chromosome inactivation and imprinting in mammals, and mating-type control in yeast. Dominant mutations that suppress or enhance PEV are thought to encode either chromatin proteins or factors that directly affect chromatin structure. We have identified an insertional mutation in Drosophila that enhances PEV and reduces transcription of the gene in the eye-antenna imaginal disc. The gene corresponds to that encoding the transcriptional regulator RPD3 in yeast, and to a human histone deacetylase. In yeast, RRD3-deletion strains show enhanced TPE, suggesting a conserved role of the histone deacetylase RPD3 in counteracting genomic silencing. This function of RPD3, which is in contrast to the general correlation between histone acetylation and increased transcription, might be due to a specialized chromatin structure at silenced loci.

Twenty seven cases of neurocysticercosis, with clinical picture of acute meningitis, are described. Twenty (74.1%) patients are male; the age was 4 to 42 years (23.6 +/- 11.7 years). The etiologic diagnosis was defined by the complement fixation test (Weinberg) and/or enzyme-linked immunosorbent assay (ELISA) for cysticercosis in the cerebrospinal fluid (c. s. f.). Six patients that realized cranial computerized tomographic scan resembling neurocysticercosis. Twenty one (77.8%) have predominance of lymphomononuclear cells in the c. s. f. obtained in the admission to the hospital; in 6 (22.2%) there were predominance of polymorphonuclear neutrophils. In this c. s. f. lymphomononuclear pleocytosis and in three that have c. s. f. neutrophil pleocytosis, suggesting the diagnosis of neurocysticercosis. The treatment of acute neurocysticercosis was made with dexamethasone. All the patients survived and were transferred to the ambulatory of Neurology for follow-up and complementary treatment.

Mutations at a few genetic loci in Drosophila cause ovarian tumors with hundreds of poorly differentiated germ cells. We examined several of these mutants to test the hypothesis that such ovarian tumors contain sex-transformed cells. By testing for expression of male germline traits, we determined that partial germline sex transformation occurs in otu, snf, Sxlfs, and bam ovarian tumors. Thus these genes are likely to be required for proper establishment of germline sexual identity.

The Drosophila melanogaster small heat-shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to alpha-crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. Two internal and adjacent deletion mutants in the conserved alpha-crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian-Virus-40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen-peroxide stresses as judged by Trypan-blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the 5' end of the conserved alpha-crystallin domain and covers the first 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N-terminal half of the conserved alpha-crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the alpha-crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus.

We used a nuclear run-on assay as a novel approach to study the changes in transcriptional activity that take place in Drosophila melanogaster during heat shock. In response to a rapid temperature upshift, total transcriptional activity in cultured KC161 cells decreased proportionally to the severity of the shock. After extended stress at 37 degrees C (15 min or more), transcription was severely reduced, and at 39 degrees C most transcription was instantaneously arrested. However, strikingly different responses were observed for individual genes. Transcription of histone H1 genes was severely inhibited even under mild heat shock conditions. Transcription of the actin 5C gene decreased progressively with increasing temperature, while transcription of the core histone genes or of the heat shock cognate genes was repressed only under severe heat shock conditions. Transcriptional activation of the D. melanogaster heat shock genes was also investigated. In unshocked cells, hsp84 was moderately transcribed, while transcriptional activity at the other protein-coding heat shock genes was undetectable (less than 0.2 polymerases per gene). Engaged but paused RNA polymerase molecules were found at the hsp70 and hsp26 genes, but not at the other heat shock genes. The rates of transcription increased with increasing temperature with a peak of expression at around 35 degrees C. At 37 degrees C, induction was less efficient, and no induction was achieved after a rapid shift to 39 degrees C. Increased transcription of the heat shock genes was observed within 1-2 min of heat shock, and maximal rates were reached within 2-5 min. Despite very similar profiles of response, different heat shock genes were transcribed at strikingly different rates, which varied over a 20-fold range. The noncoding heat shock locus 93D was transcribed at a very high rate under non-heat shock conditions, and showed a transcriptional response to elevated temperatures different from that of protein-coding heat shock genes. An estimation of the absolute rates of transcription at different temperatures was obtained.

In Drosophila melanogaster, the mechanism of sex determination is substantially different in the germ line and in the soma. In the germ line, the process is not completely cell-autonomous, but requires some signals from the soma. Only some of the genes involved in somatic sex determination are also needed for germ cell development. Recent genetic studies have identified loci required for germ-line sex determination.

Peptide methyl ketones were found to be efficient competitive inhibitors of thermitase while the chloromethyl ketone derivatives in general inhibit the enzyme irreversibly. The affinity increases by elongation of the peptide chain up to four residues indicating an interaction of the ligand with an extended binding site in thermitase. The P1 residue is phenylalanine in most of the inhibitors, according to the substrate specificity of thermitase for phenylalanine and other hydrophobic amino acids. Peptide ketones protected with the hydrophobic bencyloxycarbonyl group at the N-terminus are bound to the enzyme more tightly than the corresponding acetyl compounds. As reversible inhibitors peptide methyl ketones form a tetrahedral hemiketale with the oxygen of the active serine. In the case of the chloromethyl ketones this complex is stabilized by covalent binding to histidine-64. The inductive effect of the halogen favours the formation of the hemiketale and is expressed in about twenty times lower inhibition constants of the chloromethyl ketones. Results of the kinetic experiments show a corresponding behaviour in effector binding by thermitase and subtilisin in the S region, and from this point of view also indicate a high similarity of the active centers of both enzymes.

Gene 2, one of the seven heat shock genes from locus 67B of Drosophila melanogaster, is transcribed into two polyadenylated RNAs having different developmental profiles of expression. The smaller transcript, of about 560 nucleotides, is expressed from mid-embryogenesis to the first two larval stages and again at the beginning of pupation. The larger transcript, 780 nucleotides, contains an additional 5' exon, accounting for its larger size. It is detected in pupae and adults, is male-specific and is localized in the testes. Heat shock does not affect the abundance of these two transcripts but induces the accumulation of a third RNA species of about 2000 nucleotides. This heat-shock RNA has the same cap site as the embryonic transcript, while its 3' portion entirely includes the neighbouring hsp22 gene. It appears, therefore, that in this case, heat shock alters the normal transcription termination process. By contrast to most heat shock genes, gene 2 contains several micro introns. One long open reading frame common to the three transcripts encodes a putative polypeptide of 111 amino acid residues. No homology was found with the other small heat shock genes of locus 67B.

The low-molecular-weight heat-shock protein HSP27 is made in the absence of heat shock during Drosophila melanogaster development. An analysis of the accumulation of HSP27 during specific stages of development is presented using an antiserum recognizing this protein. Whereas HSP27 is abundant during embryogenesis, the level of this protein begins to decrease in the 20-h old embryo and is no longer detectable in second instar larvae. A high level of HSP27 is again observed in third instar larvae and reaches a maximal level in late pupae. While still abundant in young adult flies of both sexes, a greater amount of HSP27 is found in females with the protein being highly concentrated within the ovaries. Following lysis of whole pupae, about 60% of HSP27 is found in the soluble lysate fraction in a form which sediments between 5 and 20 S. Anti-HSP27 serum also recognizes three other developmentally regulated polypeptides with apparent MW of 33, 85 and 120 kDa. The 33 kDa protein accumulates in pupae while those of 85 and 120 kDa are more abundant in third instar larvae. Unlike HSP27, these proteins are not detected in embryos or ovaries. Immunoblot analysis of V8 proteolytic fragments suggests that HSP 27 and 33 kDa are related polypeptides. Exposure of the developing insect to heat-shock treatment results in increased level of HSP27. In larvae, a small amount of the 33 kDa protein accumulates following heat shock, while in pupae and adult flies a decrease in the concentration of this protein is observed after heat shock. Finally, different cellular localizations and distributions within the pupal body have been found for these developmentally regulated polypeptides.

Seven heat shock genes are clustered within 15 kilobases of DNA at the Drosophila melanogaster chromosomal site 67B. They show a complex pattern of expression in the absence of external stress during normal development of this organism. In this paper, we quantitatively compare the abundance of the messenger RNAs for these seven genes at all major stages of Drosophila development and then focus on hsp23 and hsp27 for which available antibodies allow the comparison between the accumulation of the mRNAs and that of their corresponding polypeptides. Transcripts for both genes are maximally abundant in white prepupae. We observe that the amount of hsp23 message decreases more rapidly than that of hsp27 mRNA throughout the pupal period. The maximal abundance of the proteins occurs at the middle of the pupal stage, when their corresponding RNAs have almost completely disappeared. The peaks of expression of the proteins are also broader than those of their transcripts, indicating that the half-lives of the polypeptides are longer. These observations suggest that complex mechanisms regulate the expression of the small heat shock genes during Drosophila development.

We present a detailed characterization of the structure and expression of gene 3 from the 67B locus of Drosophila melanogaster. Northern blot analysis reveals a major poly(A)+ transcript during two stages of development: mid-embryogenesis and beginning of pupation. After heat shock, the abundance of this mRNA is increased and small amounts of larger RNAs representing alternate terminations of the major transcript appear. In Schneider 3 tissue culture cells, beside the major transcript, we also observe small amounts of the larger RNAs after a heat shock. The sequencing of cDNA and genomic clones shows an intronless transcription unit with one long open reading frame. The deduced polypeptide has 169 amino acids. It shares a strong homology with the four small heat shock proteins in the region also conserved in the mammalian alpha-crystalline B2 chain. In gene 3, this homology is restricted to the first 50 residues along the conserved 83 amino acid stretch. Two heat shock regulatory elements are localized upstream from the gene.

Regional labeling of mice by injection of cytidine-3H ([3H]CR) into the footpad of the left hind leg was used to evaluate lymphocyte traffic from the left regional nodes to the right popliteal lymph node (PLN) within a 24-h period, with or without concomitant primary or secondary stimulation of the right PLN with fluid tetanus toxoid. Results indicate that 1) in the case of primary antigen injection the relative contribution of lymphocytes from the left regional nodes to the small lymphocyte population present in the stimulated right PLN 24 h after labeling was slightly, but not significantly, greater than in non-stimulated controls; 2) a booster injection of antigen into the right hind leg footpad resulted in a significantly smaller relative contribution of lymphocytes from the previously primed left regional nodes to the small lymphocyte population in the right PLN, 24 h after injection of [3H]CR and secondary stimulation, as compared with controls or animals given a primary stimulation to the receiver node; and 3) in contrast to controls and mice subjected to primary stimulation only, the right PLN 24 h after booster contained a significant number of large lymphoid cells which, or whose precursors, had migrated to this site from contralateral nodes within a day, possibly also in the form of small lymphocytes. These findings are discussed in relation to the problem of lymphocyte recruitment and divergent behavior of non-committed lymphocytes as compared with memory cells in the initial phase after primary or secondary antigenic stimulation.

An attempt was made to study the structure of the active center of thermitase by means of spin labeled peptide analogues. For this purpose, peptide methyl ketones SL-Alan-PheCH3 of different chain length (n = 0, 1, 2, 3; SL = 2,2,5,5-Tetramethyl-pyrrolin-1-oxyl-3-carbonyl) were synthesized. Synthesis and physico-chemical properties of these compounds are described and inhibition constants Ki for the interaction of these compounds with thermitase were measured. SL-Ala2-PheCH3 and SL-Ala3-PheCH3 are strong reversible inhibitors of thermitase with Ki values of 8.9 X 10(-6) M and 4.8 X 10(-7) M, respectively, whereas the analogous compounds with n = 0 and n = 1 represent only reduced affinity for this enzyme. ESR spectra of SL-Ala2-PheCH3 and SL-Ala3-PheCH3 in the presence of thermitase reveal that a great part of the SL residues of enzyme bound inhibitor molecules is not measurably restricted in their mobility whereas another part is hindered by unspecific interaction with the protein. The kinetic and ESR data are discussed with regard to the substrate binding region of the enzyme.