ABSTRACT
Prophylactic treatment with emicizumab has become an important and effective bleeding prevention for people with hemophilia A (PwHA). Perioperative management of PwHA using emicizumab prophylaxis is still challenging due to a lack of experience. Medical records of perioperative management and outcomes were reviewed, and data were collected for adult PwHA receiving emicizumab and undergoing surgical procedures between August 2019 and July 2022 at the University Medical Center Ljubljana. Twelve surgical procedures were performed in eight PwHA (one with FVIII inhibitors) while on emicizumab prophylaxis. Three minor procedures included cataract surgery, cystoscopic lithotripsy, and percutaneous coronary intervention. Nine major surgeries included four osteosyntheses, necrectomy of chronic osteomyelitis with new ankle arthrodesis, two below-knee amputations, total knee replacement, and placement of ventriculostomy after a spontaneous intraventricular hemorrhage. No major bleeds, thrombotic events or deaths, or new inhibitors appeared. Our real-world experience demonstrates that minor and major surgeries can be performed safely in PwHA on emicizumab prophylaxis. Additional data are needed to optimize dosing/duration of additional hemostatic agents in diverse invasive procedures and complex clinical situations.
ABSTRACT
An erythrocytosis is present when the red blood cell mass is increased, demonstrated as elevated hemoglobin and hematocrit in the laboratory evaluation. Congenital predispositions for erythrocytosis are rare, with germline variants in several genes involved in oxygen sensing (VHL, EGLN1, and EPAS1), signaling for hematopoietic cell maturation (EPOR and EPO), and oxygen transfer (HBB, HBA1, HBA2, and BPGM) that were already associated with the eight congenital types (ECYT1-8). Screening for variants in known congenital erythrocytosis genes with classical sequencing approach gives a correct diagnosis for only up to one-third of the patients. The genetic background of erythrocytosis is more heterogeneous, and additional genes involved in erythropoiesis and iron metabolism could have a putative effect on the development of erythrocytosis. This study aimed to detect variants in patients with yet unexplained erythrocytosis using the next-generation sequencing (NGS) approach, targeting genes associated with erythrocytosis and increased iron uptake and implementing the diagnostics of congenital erythrocytosis in Slovenia. Selected 25 patients with high hemoglobin, high hematocrit, and no acquired causes were screened for variants in the 39 candidate genes. We identified one pathogenic variant in EPAS1 gene and three novel variants with yet unknown significance in genes EPAS1, JAK2, and SH2B3. Interestingly, a high proportion of patients were heterozygous carriers for two variants in HFE gene, otherwise pathogenic for the condition of iron overload. The association between the HFE variants and the development of erythrocytosis is not clearly understood. With a targeted NGS approach, we determined an actual genetic cause for the erythrocytosis in one patient and contributed to better management of the disease for the patient and his family. The effect of variants of unknown significance on the enhanced production of red blood cells needs to be further explored with functional analysis. This study is of great significance for the improvement of diagnosis of Slovenian patients with unexplained erythrocytosis and future research on the etiology of this rare hematological disorder.
ABSTRACT
Erythrocytosis has a diverse background. While polycythaemia vera has well defined criteria, the diagnostic approach and management of other types of erythrocytosis are more challenging. The aim of study was to retrospectively analyse the aetiology and management of non-clonal erythrocytosis patients referred to a haematology outpatient clinic in an 8-year period using a 3-step algorithm. The first step was inclusion of patients with Hb > 185 g/L and/or Hct > 0.52 in men and Hb > 165 g/L and/or Hct > 0.48 in women on two visits ≥ two months apart, thus confirming true erythrocytosis. Secondly, polycythaemia vera was excluded and secondary causes of erythrocytosis (SE) identified. Thirdly, idiopathic erythrocytosis patients (IE) were referred to next-generation sequencing for possible genetic background evaluation. Of the 116 patients, 75 (65%) are men and 41 (35%) women, with non-clonal erythrocytosis 34/116 (29%) had SE, 15/116 (13%) IE and 67/116 (58%) stayed incompletely characterized (ICE). Patients with SE were significantly older and had significantly higher Hb and Hct compared to patients with IE. Most frequently, SE was attributed to obstructive sleep apnoea and smoking. Phlebotomies were performed in 56, 53 and 40% of patients in the SE, IE, and ICE group, respectively. Approx. 70% of patients in each group received aspirin. Thrombotic events were registered in 12, 20 and 15% of SE, IE and ICE patients, respectively. Congenital erythrocytosis type 4 (ECYT4) was diagnosed in one patient. The study demonstrates real-life management of non-clonal erythrocytosis which could be optimized using a 3-step diagnostic algorithm.
Subject(s)
Polycythemia/diagnosis , Polycythemia/therapy , Adult , Disease Management , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Phlebotomy , Polycythemia/congenital , Polycythemia/genetics , Retrospective StudiesABSTRACT
BACKGROUND: Erythrocytosis is a condition with an excessive number of erythrocytes, accompanied by an elevated haemoglobin and/or haematocrit value. Congenital erythrocytosis has a diverse genetic background with several genes involved in erythropoiesis. In clinical practice, nine genes are usually examined, but in approximately 70% of patients, no causative mutation can be identified. In this study, we screened 39 genes, aiming to identify potential disease-driving variants in the family with erythrocytosis of unknown cause. PATIENTS AND METHODS: Two affected family members with elevated haemoglobin and/or haematocrit and negative for acquired causes and one healthy relative from the same family were selected for molecular-genetic analysis of 24 erythrocytosis and 15 hereditary haemochromatosis-associated genes with targeted NGS. The identified variants were further analysed for pathogenicity using various bioinformatic tools and review of the literature. RESULTS: Of the 12 identified variants, two heterozygous variants, the missense variant c.471G>C (NM_022051.2) (p.(Gln157His)) in the EGLN1 gene and the intron variant c.2572-13A>G (NM_004972.3) in the JAK2 gene, were classified as low-frequency variants in European population. None of the two variants were present in a healthy family member. Variant c.2572-13A>G has potential impact on splicing by one prediction tool. CONCLUSION: For the first time, we included 39 genes in the erythrocytosis clinical panel and identified two potential disease-driving variants in the Slovene family studied. Based on the reported functional in vitro studies combined with our bioinformatics analysis, we suggest further functional analysis of variant in the JAK2 gene and evaluation of a cumulative effect of both variants.
Subject(s)
Genetic Association Studies , Genetic Predisposition to Disease , Hemochromatosis/genetics , Polycythemia/genetics , Adult , Aged , Base Sequence , Computational Biology , Family , Female , Gene Frequency/genetics , Heterozygote , Humans , Inheritance Patterns/genetics , Male , Pedigree , Polycythemia/congenital , Polymorphism, Single Nucleotide/genetics , SloveniaABSTRACT
OBJECTIVES: To evaluate the impact of the coronavirus disease 2019 (COVID-19) pandemic on acute and elective thoracic and abdominal aortic procedures. METHODS: Forty departments shared their data on acute and elective thoracic and abdominal aortic procedures between January and May 2020 and January and May 2019 in Europe, Asia and the USA. Admission rates as well as delay from onset of symptoms to referral were compared. RESULTS: No differences in the number of acute thoracic and abdominal aortic procedures were observed between 2020 and the reference period in 2019 [incidence rates ratio (IRR): 0.96, confidence interval (CI) 0.89-1.04; P = 0.39]. Also, no difference in the time interval from acute onset of symptoms to referral was recorded (<12 h 32% vs > 12 h 68% in 2020, < 12 h 34% vs > 12 h 66% in 2019 P = 0.29). Conversely, a decline of 35% in elective procedures was seen (IRR: 0.81, CI 0.76-0.87; P < 0.001) with substantial differences between countries and the most pronounced decline in Italy (-40%, P < 0.001). Interestingly, in Switzerland, an increase in the number of elective cases was observed (+35%, P = 0.02). CONCLUSIONS: There was no change in the number of acute thoracic and abdominal aortic cases and procedures during the initial wave of the COVID-19 pandemic, whereas the case load of elective operations and procedures decreased significantly. Patients with acute aortic syndromes presented despite COVID-19 and were managed according to current guidelines. Further analysis is required to prove that deferral of elective cases had no impact on premature mortality.
Subject(s)
COVID-19 , Pandemics , Asia , Elective Surgical Procedures , Europe , Humans , Italy , SARS-CoV-2 , SwitzerlandABSTRACT
BACKGROUND: A vaccine would be an ideal tool for reducing malaria's impact. PfSPZ Vaccine (radiation attenuated, aseptic, purified, cryopreserved Plasmodium falciparum [Pf] sporozoites [SPZ]) has been well tolerated and safe in >1526 malaria-naive and experienced 6-month to 65-year-olds in the United States, Europe, and Africa. When vaccine efficacy (VE) of 5 doses of 2.7 × 105 PfSPZ of PfSPZ Vaccine was assessed in adults against controlled human malaria infection (CHMI) in the United States and Tanzania and intense field transmission of heterogeneous Pf in Mali, Tanzanians had the lowest VE (20%). METHODS: To increase VE in Tanzania, we increased PfSPZ/dose (9 × 105 or 1.8 × 106) and decreased numbers of doses to 3 at 8-week intervals in a double blind, placebo-controlled trial. RESULTS: All 22 CHMIs in controls resulted in parasitemia by quantitative polymerase chain reaction. For the 9 × 105 PfSPZ group, VE was 100% (5/5) at 3 or 11 weeks (P < .000l, Barnard test, 2-tailed). For 1.8 × 106 PfSPZ, VE was 33% (2/6) at 7.5 weeks (P = .028). VE of dosage groups (100% vs 33%) was significantly different (P = .022). Volunteers underwent repeat CHMI at 37-40 weeks after last dose. 6/6 and 5/6 volunteers developed parasitemia, but time to first parasitemia was significantly longer than controls in the 9 × 105 PfSPZ group (10.89 vs 7.80 days) (P = .039), indicating a significant reduction in parasites in the liver. Antibody and T-cell responses were higher in the 1.8 × 106 PfSPZ group. CONCLUSIONS: In Tanzania, increasing the dose from 2.7 × 105 to 9 × 105 PfSPZ increased VE from 20% to 100%, but increasing to 1.8 × 106 PfSPZ significantly reduced VE. CLINICAL TRIALS REGISTRATION: NCT02613520.
Subject(s)
Malaria Vaccines , Malaria, Falciparum , Malaria , Adult , Animals , Europe , Humans , Malaria/prevention & control , Malaria, Falciparum/prevention & control , Mali , Plasmodium falciparum , Sporozoites , TanzaniaABSTRACT
The rapid and accurate diagnosis of Plasmodium falciparum malaria infection is an essential factor in malaria control. Currently, malaria diagnosis in the field depends heavily on using rapid diagnostic tests (RDTs) many of which detect circulating parasite-derived histidine-rich protein 2 antigen (PfHRP2) in capillary blood. P. falciparum strains lacking PfHRP2, due to pfhrp2 gene deletions, are an emerging threat to malaria control programs. The novel assay described here, named qHRP2/3-del, is well suited for high-throughput screening of P. falciparum isolates to identify these gene deletions. The qHRP2/3-del assay identified pfhrp2 and pfhrp3 deletion status correctly in 93.4% of samples with parasitemia levels higher than 5 parasites/µL when compared to nested PCR. The qHRP2/3-del assay can correctly identify pfhrp2 and pfhrp3 gene deletions in multiple strain co-infections, particularly prevalent in Sub-Saharan countries. Deployment of this qHRP2/3-del assay will provide rapid insight into the prevalence and potential spread of P. falciparum isolates that escape surveillance by RDTs.
Subject(s)
Antigens, Protozoan/genetics , Antigens, Protozoan/metabolism , Gene Deletion , Plasmodium falciparum/genetics , Polymerase Chain Reaction/methods , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Plasmodium falciparum/physiologyABSTRACT
In 2016, there were more cases and deaths caused by malaria globally than in 2015. An effective vaccine would be an ideal additional tool for reducing malaria's impact. Sanaria® PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) has been well tolerated and safe in malaria-naïve and experienced adults in the United States and Mali and protective against controlled human malaria infection with Pf in the United States and field transmission of Pf in Mali, but had not been assessed in younger age groups. We, therefore, evaluated PfSPZ Vaccine in 93 Tanzanians aged 45 years to 6 months in a randomized, double-blind, normal saline placebo-controlled trial. There were no significant differences in adverse events between vaccinees and controls or between dosage regimens. Because all age groups received three doses of 9.0 × 105 PfSPZ of PfSPZ Vaccine, immune responses were compared at this dosage. Median antibody responses against Pf circumsporozoite protein and PfSPZ were highest in infants and lowest in adults. T-cell responses were highest in 6-10-year olds after one dose and 1-5-year olds after three doses; infants had no significant positive T-cell responses. The safety data were used to support initiation of trials in > 300 infants in Kenya and Equatorial Guinea. Because PfSPZ Vaccine-induced protection is thought to be mediated by T cells, the T-cell data suggest PfSPZ Vaccine may be more protective in children than in adults, whereas infants may not be immunologically mature enough to respond to the PfSPZ Vaccine immunization regimen assessed.
Subject(s)
Antibodies, Protozoan/blood , Malaria Vaccines/immunology , Malaria, Falciparum/prevention & control , Plasmodium falciparum/immunology , T-Lymphocytes/physiology , Adolescent , Adult , Antibody Formation , Child , Child, Preschool , Double-Blind Method , Female , Humans , Infant , Malaria Vaccines/adverse effects , Male , Middle Aged , Tanzania , Vaccines, AttenuatedABSTRACT
The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptome analysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem-/- mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect of CREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with its impact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated in KO testis and are therefore possible novel targets of CREM.
Subject(s)
Cyclic AMP Response Element Modulator/physiology , Gene Expression Regulation , Testis/metabolism , Animals , Apoptosis , Biological Transport , Cyclic AMP Response Element Modulator/biosynthesis , Fertilization , Gene Expression Profiling , Homozygote , Male , Melatonin/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction/methods , Spermatids/metabolism , Spermatogenesis , Spermatozoa/metabolismABSTRACT
PURPOSES: The study sets the stage for temporal information processing as a fundamental basis of human cognition and a novel neurorehabilitation method. We focus on auditory perception of temporal order and address the following questions: (1) do subjects' age, gender, hearing status and cognitive functioning influence temporal ordering abilities; (2) are there any differences between Polish and German subjects on these abilities? METHODS: 86 Polish and 82 German subjects aged from 20 to 69 years were classified into 5 age groups. Subjects identified the order of two 1-ms clicks presented monaurally in rapid succession. The temporal order threshold (i.e. the minimum temporal gap required to report the stimulus order at 75% correctness) was assessed for each individual. RESULTS: There were no differences between Polish and German subjects on temporal ordering. In both samples, a significant prolongation of temporal-order threshold was observed in subjects older than 60 years of age. Temporal processing was relatively resistant to subjects' hearing status, but it depended on cognitive competence. Thus, it is not chronological age as such but cognitive competence that may explain age-related decreases of temporal acuity. Furthermore, potential effects of age or gender are robust against the language background as no differences were observed between Polish and German subjects. CONCLUSIONS: We propose the existence of a neural mechanism underlying the perception of rapid changes in non-verbal acoustic features which constitute a frame for speech perception in many languages. This finding may be important with respect to future applications of temporal training in speech therapy programs designed for patients with receptive language disorders of different etiologies.
Subject(s)
Aging/physiology , Auditory Perception/physiology , Language Therapy/methods , Linguistics/methods , Multilingualism , Acoustic Stimulation/methods , Adult , Aged , Female , Humans , Language , Male , Middle Aged , Psychomotor Performance/physiology , Time Factors , Young AdultABSTRACT
BACKGROUND: Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration. RESULTS: In the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons. CONCLUSIONS: Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.
Subject(s)
Circadian Rhythm/genetics , Gene Expression , Mice, Inbred Strains/genetics , Polymerase Chain Reaction , Animals , Humans , Mice , Molecular Sequence Data , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/standardsABSTRACT
The present paper investigates the effects of age, sex, and cognitive factors on temporal-order perception. Nine temporal-order tasks were employed using two and four stimuli presented in the auditory and visual modalities. Significantly increased temporal-order thresholds (TOT) in the elderly were found for almost all tasks, while sex differences were only observed for two tasks. Multiple regression analyses show that the performance on most temporal-order tasks can be predicted by cognitive factors, such as speed of fluid reasoning, short-term memory, and attention. However, age was a significant predictor of TOT in three tasks using visual stimuli. We conclude (1) that age-related differences can often be attributed to cognitive factors involved in temporal-order perception, and (2) that the concept of temporal-order perception is more complex than implied by the current models.
Subject(s)
Aging/psychology , Cognition , Sex Characteristics , Time Perception , Acoustic Stimulation , Adult , Aged , Aged, 80 and over , Analysis of Variance , Female , Humans , Male , Middle Aged , Photic Stimulation , Regression Analysis , Young AdultABSTRACT
We show for the first time that isoforms of the cAMP response element modulator Crem, regulate the circadian expression of Cyp51 and other cholesterogenic genes in the mouse liver. In the wild type mice the expression of Cyp51, Hmgs, Fpps, and Sqs is minimal between CT12 and CT16 and peaks between CT20 and CT24. Cyp51, Fpps, and Sqs lost the circadian behavior in Crem-/- livers while Hmgcr is phase advanced from CT20 to CT12. This coincides with a phase advance of lathosterol/cholesterol ratio, as detected by GC-MS. Overexpression of CREMtau and ICER has little effect on the Hmgcr proximal promoter while they influence expression from the CYP51 promoter. Our data indicate that Crem-dependent regulation of Cyp51 in the liver results in circadian expression of this gene. We propose that cAMP signaling might generally be involved in the circadian regulation of cholesterol synthesis on the periphery.
Subject(s)
Cholesterol/biosynthesis , Circadian Rhythm , Cyclic AMP Response Element Modulator/metabolism , Cytochrome P-450 Enzyme System/biosynthesis , Hydroxymethylglutaryl-CoA Synthase/biosynthesis , Liver/metabolism , Animals , Circadian Rhythm/genetics , Cyclic AMP Response Element Modulator/genetics , Gene Expression Regulation , Mice , Mice, Knockout , Promoter Regions, Genetic , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sterol 14-DemethylaseABSTRACT
Neuropsychological studies in brain-injured patients with aphasia and children with specific language-learning deficits have shown the dependence of language comprehension on auditory processing abilities, i.e. the detection of temporal order. An impairment of temporal-order perception can be simulated by time reversing segments of the speech signal. In our study, we investigated how different lengths of time-reversed segments in speech influenced comprehension in ten native German speakers and ten participants who had acquired German as a second language. Results show that native speakers were still able to understand the distorted speech at segment lengths of 50 ms, whereas non-native speakers only could identify sentences with reversed intervals of 32 ms duration. These differences in performance can be interpreted by different levels of semantic and lexical proficiency. Our method of temporally-distorted speech offers a new approach to assess language skills that indirectly taps into lexical and semantic competence of non-native speakers.
Subject(s)
Comprehension/physiology , Language , Speech Perception/physiology , Verbal Behavior/physiology , Acoustic Stimulation/methods , Adult , Female , Humans , Male , Middle Aged , Statistics, Nonparametric , Time FactorsABSTRACT
Some authors have suggested separate mechanisms for the processing of temporal intervals above versus below 2-3s. Given that the evidence is mixed, the present experiment was carried out as a critical test of the separate-mechanism hypothesis. Subjects reproduced five standard durations of 1-5s presented in the auditory and visual modalities. The Corsi-block test was used to assess effects of working-memory span on different interval lengths. Greater working-memory span was associated with longer reproductions of intervals of 3-5s. A factor analysis run on mean reproduced intervals revealed one modality-unspecific factor for durations of 1-2s and two modality-specific factors for longer intervals. These results are interpreted as further indications that two different processes underlie temporal reproductions of shorter and longer intervals.
Subject(s)
Memory, Short-Term/physiology , Mental Processes/physiology , Time Perception/physiology , Acoustic Stimulation/methods , Adult , Age Distribution , Aged , Aged, 80 and over , Analysis of Variance , Auditory Perception/physiology , Factor Analysis, Statistical , Female , Humans , Male , Middle Aged , Photic Stimulation/methods , Task Performance and Analysis , Visual Perception/physiologyABSTRACT
Standard diagnostic procedures for assessing temporal-processing abilities of adult patients with aphasia have so far not been developed. In our study, temporal-order measurements were conducted using two different experimental procedures to identify a suitable measure for clinical studies. Additionally, phoneme-discrimination abilities were tested on the word, as well as on the sentence level, as a relationship between temporal processing and phoneme-discrimination abilities is assumed. Patients with aphasia displayed significantly higher temporal-order thresholds than control subjects. The detection of an association between temporal processing and speech processing, however, depended on the stimuli and the phoneme-discrimination tasks used. Our results also suggest top-down feedback on phonemic processing.
Subject(s)
Aphasia/physiopathology , Auditory Perception/physiology , Comprehension , Discrimination, Psychological , Adult , Aged , Aged, 80 and over , Case-Control Studies , Comprehension/physiology , Discrimination, Psychological/physiology , Female , Humans , Language , Male , Middle Aged , Multivariate Analysis , Time FactorsABSTRACT
To determine the relative safety of onboard display positions while driving, participants performed a lane-keeping task in a driving simulator. Concurrently, they reacted to a light by pushing the brake pedal. A secondary task was projected onto a display at one of the seven different locations in the cockpit. Behavioral data, eye movements, and subjective rating scales showed that the manipulation of display information during driving disturbed drivers' performance exponentially as a function of distance between the line of sight to the outside primary task and the onboard display position. Vertical eccentricity had a greater detrimental effect than horizontal distance. Under a certain condition with a high secondary task load, reaction time of pushing the brake to the outside stimulus nearly doubled with a diagonal eccentricity of 35 degrees as compared to lower eccentricities. Subjective workload measures complement the behavioral data of clear detrimental effects with eccentricities of at least 35 degrees .
Subject(s)
Automobile Driving , Computer Simulation , Data Display , User-Computer Interface , Ergonomics , Humans , Japan , SafetyABSTRACT
Lanosterol 14alpha-demethylase (CYP51) responds to cholesterol feedback regulation through sterol regulatory element binding proteins (SREBPs). The proximal promoter of CYP51 contains a conserved region with clustered regulatory elements: GC box, cAMP-response elements (CRE-like), and sterol regulatory element (SRE). In lipid-rich (SREBP-poor) conditions, the CYP51 mRNA drops gradually, the promoter activity is diminished, and no DNA-protein complex is observed at the CYP51-SRE1 site. The majority of cAMP-dependent transactivation is mediated through a single CRE (CYP51-CRE2). Exposure of JEG-3 cells to forskolin, a mediator of the cAMP-dependent signaling pathway, provokes an immediate early response of CYP51, which has not been described before for any cholesterogenic gene. The CYP51 mRNA increases up to 4-fold in 2 h and drops to basal level after 4 h. The inducible cAMP early repressor (ICER) is involved in attenuation of transcription. Overexpressed CRE-binding protein (CREB)/CRE modulator (CREM) transactivates the mouse/human CYP51 promoters containing CYP51-CRE2 independently of SREBPs, and ICER decreases the CREB-induced transcription. Besides the increased CYP51 mRNA, forskolin affects the de novo sterol biosynthesis in JEG-3 cells. An increased consumption of lanosterol, a substrate of CYP51, is observed together with modulation of the postlanosterol cholesterogenesis, indicating that cAMP-dependent stimuli cross-talk with cholesterol feedback regulation. CRE-2 is essential for cAMP-dependent transactivation, whereas SRE seems to be less important. Interestingly, when CREB is not limiting, the increasing amounts of SREBP-1a fail to transactivate the CYP51 promoter above the CREB-only level, suggesting that hormones might have an important role in regulating cholesterogenesis in vivo.
Subject(s)
Cholesterol/biosynthesis , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Genes, Immediate-Early , Oxidoreductases/genetics , Oxidoreductases/metabolism , Animals , Cell Line, Tumor , Colforsin/pharmacology , Cyclic AMP/genetics , Cyclic AMP/metabolism , Cyclic AMP/physiology , Humans , Lipids/antagonists & inhibitors , Mammals/genetics , Mice , Promoter Regions, Genetic , RNA, Messenger/metabolism , Response Elements , Signal Transduction/drug effects , Sterol 14-Demethylase , Sterols/metabolism , Time Factors , Transcriptional ActivationABSTRACT
Meiosis activating sterols (MAS) are biologically active post-lanosterol intermediates of cholesterol biosynthesis that are synthetized primarily in the gonads, including the sperm. MAS reinitiate the meiosis of oocytes in vitro while in vivo they seem to contribute to the oocyte quality and the progression of meiosis. The mRNAs for the MAS-producing enzyme lanosterol 14alpha-demethylase (CYP51) arise by alternative poly (A) signal selection. Only signals with low cleavage activity are used in the testis. Translation of mammalian CYP51s starts at one of the tandem in-frame ATGs. CYP51 protein of the bull is shorter compared to the human due to the usage of a more downstream translation start site. CYP51 proteins are post-translationally modified by glycosylations in the Golgi and on acrosomal membranes of the sperm. Green fluorescence protein-based ex vivo system has been developed to aid studying the intracellular transport of the MAS-producing CYP51. The influence of the post-translational modifications on MAS-synthesizing capacity is under investigation.