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The gene for autosomal recessive retinitis pigmentosa (RP12) with preserved para-arteriolar retinal pigment epithelium was previously mapped close to the F13B gene in region 1q31→q32.1. A 4-Mb yeast artificial chromosome contig spanning this interval was constructed to facilitate cloning of the RP12 gene. The contig comprises 25 sequence-tagged sites, polymorphic markers, and single-copy probes, including five newly obtained probes. The contig orders the F13B and HF1 genes, as well as five expressed sequence tags, with respect to the integrated genetic map of this region. Homozygosity mapping resulted in refinement of the candidate gene locus for RP12 to a 1.3-cM region. Currently, approximately 1 Mb of the contig is represented in P1-derived artificial chromosome (PAC) clones. Direct screening of a cDNA library derived from neural retina with PACs resulted in identification of the human elongation factor 1α pseudogene (EEF1AL11) and a human ribosomal protein L30 pseudogene (RPL30). A physical and genetic map covering the entire RP12 candidate gene region was constructed. [ABSTRACT FROM AUTHOR]

Aim: Blood pressure (BP) responses to exercise are frequently measured, with the concern that greater increases are a marker of disease. We sought to characterize the normal exercise BP response in healthy adults and its relationships with age, sex, and fitness., Methods: 589 participants (median age 46 [IQR 24-56] years, 81% male) underwent cardiopulmonary exercise testing with repeated, automated BP measures. An exaggerated maximal systolic BP (SBPmax) was defined from current guidelines as ≥210 mmHg in males and ≥190 mmHg in females. Individual linear regression analyses defined the relationship between BP and workload (SBP/W-slope and DBP/W-slope). Participants with or without an exaggerated SBPmax and above or below median SBP/W-slope were compared., Results: An exaggerated SBPmax was found in 51% of males and 64% of females and was more prevalent in endurance-trained athletes (males 58%, females 72%, p<0.001). The mean SBP/W-slope was lower in males (0.24±0.10 mmHg/W) than females (0.27±0.12 mmHg/W), p=0.031. In both sexes, peak oxygen uptake (VO2peak) was inversely correlated with SBP/W-slope (p<0.01). Those with an exaggerated SBPmax and below-median SBP/W-slope were 10 years younger and had a 20% higher VO2peak, on average (p<0.001). A non-exaggerated SBPmax and above-median SBP/W-slope was observed in older individuals with the lowest VO2peak., Conclusion: In a large cohort of healthy individuals, an exaggerated SBPmax was common and associated with higher fitness. In contrast, higher SBP indexed to workload was associated with older age, lower fitness, and female sex. Thus, sex, age and fitness should be considered when evaluating BP response to exercise., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Fragmented QRS (fQRS) on a 12-lead ECG has been linked with adverse outcome. However, the visual scoring of ECGs is prone to inter- and intra-observer variability., Methods: Five observers, two experienced and three novel, assessed fQRS in 712 digital ECGs, 100 were re-evaluated to assess intra-observer variability. Fleiss and Cohen's Kappa were calculated and compared between subgroups., Results: The inter-observer variability for assessing fQRS in all leads combined was substantial with a Kappa of 0.651. Experienced observers only had a better agreement with a Kappa of 0.823. Intra-observer variability ranged from 0.736 to 0.880. In the subgroup with ventricular pacing the inter-observer variability was even significantly larger when compared to ECGs with normal QRS duration (Kappa 0.493 vs 0.664, p<0.001)., Conclusion: The visual assessment of QRS fragmentation is prone to inter- and intra-observer variability, mainly influenced by the experience of the observers, the underlying rhythm and QRS morphology., (Copyright © 2017. Published by Elsevier Inc.)

The functional characterization of the Halloween genes represented a major breakthrough in the elucidation of the ecdysteroid biosynthetic pathway. These genes encode cytochrome P450 enzymes catalyzing the final steps of ecdysteroid biosynthesis in the dipteran Drosophila melanogaster and the Lepidoptera Manduca sexta and Bombyx mori. This is the first report on the identification of two Halloween genes, spook (spo) and phantom (phm), from a hemimetabolous orthopteran insect, the desert locust Schistocerca gregaria. Using q-RT-PCR, their spatial and temporal transcript profiles were analyzed in both final larval stage and adult locusts. The circulating ecdysteroid titers in the hemolymph were measured and found to correlate well with changes in the temporal transcript profiles of spo and phm. Moreover, an RNA interference (RNAi)-based approach was employed to study knockdown effects upon silencing of both transcripts in the fifth larval stage. Circulating ecdysteroid levels were found to be significantly reduced upon dsRNA treatment., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Information on the structural characteristics and inhibitory activity of the pacifastin family is restricted to a handful of locust pacifastin-related inhibitors. In this report the optimization of a bacterial recombinant expression system is described, resulting in the high yield production of pacifastin-like inhibitors of the desert locust. Subsequently, the relative inhibitory activity of these peptides towards mammalian, locust and caterpillar digestive peptidases has been compared. In general, the enzyme specificity of locust pacifastin-like inhibitors towards trypsin- or chymotrypsin-like peptidases corresponds to the nature of the P1-residue at the reactive site. In addition, other structural characteristics, including specific core interactions, have been reported to result in a different affinity of pacifastin members towards digestive trypsin-like enzymes from mammals and arthropods. One remarkable observation in this study is a specifically designed pacifastin-like peptidase inhibitor, which, unlike other inhibitors of the same family, does not display this specificity and selectivity towards digestive enzymes from different animals., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Male insects change behaviors of female partners by co-transferring accessory gland proteins (Acps) like sex peptide (SP), with their sperm. The Drosophila sex peptide receptor (SPR) is a G protein-coupled receptor expressed in the female's nervous system and genital tract. While most Acps show a fast rate of evolution, SPRs are highly conserved in insects. We report activation of SPRs by evolutionary conserved myoinhibiting peptides (MIPs). Structural determinants in SP and MIPs responsible for this dual receptor activation are characterized. Drosophila SPR is also expressed in embryonic and larval stages and in the adult male nervous system, whereas SP expression is restricted to the male reproductive system. MIP transcripts occur in male and female central nervous system, possibly acting as endogenous SPR ligands. Evolutionary consequences of the promiscuous nature of SPRs are discussed. MIPs likely function as ancestral ligands of SPRs and could place evolutionary constraints on the MIP/SPR class.

Members of the pacifastin family have been characterized as serine peptidase inhibitors (PI), but their target enzyme(s) are unknown in insects. So far, the structural and biochemical characteristics of pacifastin-like PI have only been studied in locusts. Here we report the molecular identification and functional characterization of a pacifastin-like precursor in a lepidopteran insect, i.e. the silkworm Bombyx mori. The bmpp-1 gene contains 17 exons and codes for two pacifastin-related precursors of different length. The longest splice variant encodes 13 inhibitor domains, more than any other pacifastin-like precursor in arthropods. The second transcript lacks two exons and codes for 11 inhibitor domains. By studying the expression profile of the Bombyx pacifastin-like gene a different expression pattern for the two variants was observed suggesting functional diversification. Next, several PI domains of BMPP-1 were produced and, contrary to locust pacifastin peptides, they were found to be potent inhibitors of both bovine trypsin and chymotrypsin. Surprisingly, the same Bombyx PI are only weak inhibitors of endogenous digestive peptidases, indicating that other peptidases are the in vivo targets. Interestingly, the Bombyx PI inhibit a fungal trypsin-like cuticle degrading enzyme, suggesting a protective function for BMPP-1 against entomopathogenic fungi.

Background: To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of these genes., Results: We have identified seven orthologs of commonly used housekeeping genes in the desert locust. The selected genes were the orthologs of actin, EF1a, GAPDH, RP49, TubA1, Ubi, and CG13220. By employing real time RT-PCR we have analysed the expression of these housekeeping genes in brain tissue of fifth instar nymphs and adults. In the brain of fifth instar nymphs geNorm indicated Sg-EF1a, Sg-GAPDH and Sg-RP49 as most stable genes, while Normfinder ranked Sg-RP49, Sg-EF1a and Sg-ACT as most suitable candidates for normalization. The best normalization candidates for gene expression studies in the brains of adult locusts were Sg-EF1a, Sg-GAPDH and Sg-Ubi according to geNorm, while Normfinder determined Sg-GAPDH, Sg-Ubi and Sg-ACT as the most stable housekeeping genes., Conclusion: To perform transcript profiling studies on brains of the desert locust, the use of Sg-RP49, Sg-EF1a and Sg-ACT as reference genes is proposed for studies of fifth instar nymphs. In experiments with adult brains, however, the most preferred reference genes were Sg-GAPDH, Sg-Ubi and Sg-EF1a. These data will facilitate transcript profiling studies in desert locusts and provide a good starting point for the initial selection of genes for validation studies in other insects.

Background: Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. Structural and biochemical characteristics of insect pacifastin-like peptides have been studied intensively, but only one inhibitor has been functionally characterised. Recent sequencing projects of metazoan genomes have created an unprecedented opportunity to explore the distribution, evolution and functional diversification of pacifastin genes in the animal kingdom., Results: A large scale in silico data mining search led to the identification of 83 pacifastin members with 284 inhibitor domains, distributed over 55 species from three metazoan phyla. In contrast to previous assumptions, members of this family were also found in other phyla than Arthropoda, including the sister phylum Onychophora and the 'primitive', non-bilaterian Placozoa. In Arthropoda, pacifastin members were found to be distributed among insect families of nearly all insect orders and for the first time also among crustacean species other than crayfish and the Chinese mitten crab. Contrary to precursors from Crustacea, the majority of insect pacifastin members contain dibasic cleavage sites, indicative for posttranslational processing into numerous inhibitor peptides. Whereas some insect species have lost the pacifastin gene, others were found to have several (often clustered) paralogous genes. Amino acids corresponding to the reactive site or involved in the folding of the inhibitor domain were analysed as a basis for the biochemical properties., Conclusion: The absence of the pacifastin gene in some insect genomes and the extensive gene expansion in other insects are indicative for the rapid (adaptive) evolution of this gene family. In addition, differential processing mechanisms and a high variability in the reactive site residues and the inner core interactions contribute to a broad functional diversification of inhibitor peptides, indicating wide ranging roles in different physiological processes. Based on the observation of a pacifastin gene in Placozoa, it can be hypothesized that the ancestral pacifastin gene has occurred before the divergence of bilaterian animals. However, considering differences in gene structure between the placozoan and other pacifastin genes and the existence of a 'pacifastin gene gap' between Placozoa and Onychophora/Arthropoda, it cannot be excluded that the pacifastin signature originated twice by convergent evolution.

Background: To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63-78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software., Results: In total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover., Conclusion: Our study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for candidate retinal disease gene identification or gene dose-dependent therapeutic studies.

Members of the pacifastin family are serine peptidase inhibitors, found in arthropods and have many members within different insect orders. Based on their structural characteristics, inhibitors of this peptide family are divided into two groups (I and II). Members of both groups exhibit specificity towards different types of serine peptidases. In addition, group I inhibitors display species selectivity. The specificity and selectivity of these inhibitors depends on the nature of their P1 residue and on additional interaction sites at the inhibitor's surface. Functional analysis studies have shown that crustacean pacifastin plays a key role in the immune response, whereas insect pacifastin-like peptides have multiple regulatory functions in processes involved in immunity, reproduction, phase transition, etc.

The expression pattern of functional members of the metallothionein (MT) gene family was studied in the haematopoietic precursor cell lines, K562, DAMI, MEG-01, and ELF-153 in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 with 10% (v/v) foetal calf serum, with or without different zinc supplements. Expression of MT isogenes was analysed by quantitative real-time PCR (RT-PCR) using mRNA extracted from cultured cells. The more mature K562, DAMI, and MEG-01 cell lines exhibited transcription of all MT isogenes, except MT-3 and MT-4. Relative quantitative expression of MT isogenes in the mature cell lines such as K562, DAMI, and MEG-01 was higher than in the immature ELF-153 cell line. Immunohistochemical staining (IHC) reveals an increased MT protein biosynthesis in more mature cell lines such as K562, DAMI and MEG-01 greater than in the immature ELF-153 cell line. Real-time PCR and immunohistochemical staining for investigating the effect of phorbol ester and hemin (haematopoietic differentiation stimuli) on expression of MT isogenes in K562 cells reveals that phorbol ester induces increased MT transcription and biosynthesis. Therefore, to our knowledge, the role of MT in differentiation in human haematopoietic precursor cell lines is here reported for the first time.

Members of the insulin superfamily are not restricted to vertebrates, but have also been identified in invertebrate species. In the current report, we present the characterization of Scg-insulin-related peptide (IRP), an insulin-related peptide in the desert locust, Schistocerca gregaria. This peptide was isolated from corpora cardiaca (CC) extracts by means of a high-performance liquid chromatography (HPLC)-based purification strategy. Subsequent cloning and sequencing of the corresponding cDNA revealed that the encoded Scg-IRP precursor displays the structural organization that is typical for members of the insulin superfamily. Moreover, immunocytochemistry on brain tissue sections demonstrated the presence of Scg-IRP in median neurosecretory cells of the pars intercerebralis and their projections towards the storage part of the CC. Quantitative real-time RT-PCR studies revealed the presence of Scg-IRP transcripts in a variety of tissues, including nervous tissue and fat body. Furthermore, these transcripts showed a tissue- and phase-dependent, temporal regulation during the reproductive cycle of adult males and females. Finally, we demonstrated that Scg-IRP interacts in vitro with a recombinant neuroparsin, a locust protein displaying sequence similarity with vertebrate IGF binding proteins.

The prophenoloxidase-activating system is an important component of the innate immune response of insects, involved in wound healing and melanotic encapsulation. In this paper we show that in the desert locust, Schistocerca gregaria, hemocytes, challenged with microbial elicitors, are indispensable for the limited proteolytic activation of prophenoloxidase (proPO) in plasma. In addition, we assessed the influence of serine protease inhibitors on the induction of PO-activity in plasma. While soybean Bowman-Birk inhibitor (SBBI) inhibited the PO activation by laminarin-treated hemocytes, the endogenous pacifastin-related inhibitors, SGPI-1 (S. gregaria pacifastin-related inhibitor-1) and SGPI-2 did not affect the PO-activity under similar conditions. On the other hand, real-time PCR analysis revealed that the transcripts, encoding SGPI-1-3, were more abundant in the fat body of immune challenged animals, as compared to control animals.

Purpose: To determine whether variants in the candidate genes TLR4, CCL2, and CCR2 are associated with age-related macular degeneration (AMD)., Methods: This study was performed in two independent Caucasian populations that included 357 cases and 173 controls from the Netherlands and 368 cases and 368 controls from the United States. Exon 4 of the TLR4 gene and the promoter, all exons, and flanking intronic regions of the CCL2 and CCR2 genes were analyzed in the Dutch study and common variants were validated in the U.S. study. Quantitative (q)PCR reactions were performed to evaluate expression of these genes in laser-dissected retinal pigment epithelium from 13 donor AMD and 13 control eyes., Results: Analysis of single nucleotide polymorphisms (SNPs) in the TLR4 gene did not show a significant association between D299G or T399I and AMD, nor did haplotypes containing these variants. Univariate analyses of the SNPs in CCL2 and CCR2 did not demonstrate an association with AMD. For CCR2, haplotype frequencies were not significantly different between cases and controls. For CCL2, one haplotype containing the minor allele of C35C was significantly associated with AMD (P = 0.03), but this did not sustain after adjustment for multiple testing (q = 0.30). Expression analysis did not demonstrate altered RNA expression of CCL2 and CCR2 in the retinal pigment epithelium from AMD eyes (for CCL2 P = 0.62; for CCR2 P = 0.97)., Conclusions: No evidence was found of an association between TLR4, CCR2, and CCL2 and AMD, which implies that the common genetic variation in these genes does not play a significant role in the etiology of AMD.

Purpose: To obtain and analyze the gene expression changes after ischemic preconditioning (IPC) in the rat retina., Methods: Ischemic damage to the inner retina can be prevented by a short, non-deleterious, ischemic insult of 5 min applied 24 h preceding a full ischemic insult of 60 min; a phenomenon termed tolerance or IPC. The time course of changes in gene expression after induction of IPC was assessed by 22K oligonucleotide microarrays, followed by real-time quantitative polymerase chain reaction (qPCR) validation. Functional pathways of interest were identified by Gene Ontology-term analysis., Results: Histology confirmed that IPC induction by 5 min of retinal ischemia results in a complete protection against the neurodegenerative effects of a 60 min ischemic period applied 24 or 48 h later. The microarray analysis revealed differential expression of 104 known genes at one or more time points between 1 h and 7 days after IPC. The group of altered genes contained a significant overrepresentation of genes involved in aminoacyl-tRNA synthetase activity (Iars, Lars, Cars, Yars, Gars, Tars), amino acid transport (Slc3a2, Slc6a6, Slc7a1, Slc38a2), regulation of transcription (including Egr1, Egr4, Nr4a1, Nr4a3, c-fos), and cell death (including Anxa1, Trib3). qPCR assays on cDNA of individual animals confirmed the microarray results., Conclusions: Endogenous neuroprotection, provoked by ischemic preconditioning is associated with changes in transcript levels of several functionally-related groups of genes. During the time window of effective protection, transcript levels of genes encoding for aminoacyl-tRNA synthetases and for amino acid transport are reduced. These changes suggest that a reduction of translational activity may play a significant role in preconditioning-mediated neuroprotection.

A major unresolved issue in insect endocrinology concerns the question of whether or not insects have sex hormones. Conclusive evidence in favor of the presence of such hormones awaits the establishment of appropriate bioassays in males. The cuticle of sexually mature males of the desert locust Schistocerca gregaria turns yellow in gregarious conditions only. Neither females nor isolated males ever turn yellow. The yellowing is due to the deposition in the cuticle of a male-specific Yellow Protein (YP), of which the amino acid sequence is known. In this paper, we describe the partial cloning of the cDNA encoding this Yellow Protein. The tissue distribution and temporal expression of the YP-mRNA is studied in detail using RT-PCR. Furthermore, an RT-PCR based bioassay was developed, which may serve as a reliable tool to help identify the hormones controlling the yellowing process. In addition to juvenile hormone, we have shown that a factor present in the brain-corpora cardiaca is involved in the yellow coloration, as injection of an extract induces the expression of YP-mRNA in isolated gregarious males.