Acute mastoiditis in children: A tertiary care center experience in 2015-2021.

Background: Acute mastoiditis is a suppurative infection of mastoid air cells and is the most common intratemporal complication of otitis media. Aim: This study aimed to evaluate the demographic and clinical characteristics and treatment outcomes of children with acute mastoiditis (AM). Patients and Methods: We retrospectively reviewed the medical records of hospitalized pediatric patients aged between 1 month and 18 years with a diagnosis of AM between May 2015 and December 2021. Results: A total of 28 hospitalized children with AM were enrolled in this study, of whom 22 (78.6%) were males and 6 (21.4%) were females with a mean ± standard deviation age of 93.5 ± 53.2 months (range = 6 months-16.1 years). The most common clinical symptoms were postauricular erythema (n = 17, 60.7%), tenderness (n = 16, 57.1%), swelling (n = 14, 50%), fever (n = 14, 50%), and auricular protrusion (n = 7, 25%). Mastoiditis complications occurred in 10 (35.7%) children. The most common extracranial complication was subperiosteal abscess (n = 8, 28.6%). The erythrocyte sedimentation rate (ESR) and the rate of antibiotic use before hospitalization were higher in patients with complicated mastoiditis (P = 0.006 and P = 0.039, respectively). Surgery was performed in 12 (42.9%) patients. Statistically, more surgical interventions were performed in patients who developed complications (P = 0.003). Conclusion: AM continues as an important disease of childhood. Successful results are obtained with systemic antibiotic therapy and additional surgical intervention as necessary. A careful evaluation of patients with a high ESR and those who received antibiotic therapy before hospitalization is appropriate due to the correlation between these factors and the risk of complication development.

Klf9 is necessary and sufficient for Purkinje cell survival in organotypic culture.

During their phase of developmental programmed cell death (PCD), neurons depend on target-released trophic factors for survival. After this period, however, they critically change as their survival becomes target-independent. The molecular mechanisms underlying this major transition remain poorly understood. Here, we investigated, which transcription factors (TFs) might be responsible for the closure of PCD. We used Purkinje cells as a model since their PCD is restricted to the first postnatal week in the mouse cerebellum. Transcriptome analysis of Purkinje cells during or after PCD allowed the identification of Krüppel like factor 9 (Klf9) as a candidate for PCD closure, given its high increase of expression at the end of the 1st postnatal week. Klf9 function was tested in organotypic cultures, through lentiviral vector-mediated manipulation of Klf9 expression. In absence of trophic factors, the Purkinje cell survival rate is of 40%. Overexpression of Klf9 during PCD dramatically increases the Purkinje cell survival rate from 40% to 88%, whereas its down-regulation decreases it to 14%. Accordingly, in organotypic cultures of Klf9 knockout animals, Purkinje cell survival rate is reduced by half as compared to wild-type mice. Furthermore, the absence of Klf9 could be rescued by Purkinje cell trophic factors, Insulin growth factor-1 and Neurotrophin3. Altogether, our results ascribe a clear role of Klf9 in Purkinje cell survival. Thus, we propose that Klf9 might be a key molecule involved in turning off the phase of Purkinje PCD.

Experimental Lactococcus garvieae infection in rainbow trout, Oncorhynchus mykiss, Walbaum 1792: a comparative histopathological and immunohistochemical study.

The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young-Adult IP groups and Young-Adult IM groups, each contain 30 fish) and four control groups (Young-Adult IP Control groups and Young-Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976. Fish in the IP groups were intraperitoneally administered an inoculate containing 10(6) cfu mL(-1) bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10(8) cfu mL(-1) bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin-biotin-peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.

Pathomorphological, immunohistochemical and bacteriological findings in budgerigars (Melopsittacus undulatus) naturally infected with S. Gallinarum.

The present study describes the pathological and bacteriological findings and diagnosis by immunoperoxidase and immunofluorescence methods in budgerigars (Melopsittacus undulatus) naturally infected with Salmonella gallinarum obtained from three commercial budgerigar rearing farms. The course of the disease in young budgerigars was peracute or acute, whereas in adult budgerigars the disease was acute or chronic. Clinically, yellow-white diarrhoea was observed in the young budgerigars with the acute form. In the adult budgerigars with the acute and chronic forms, a decrease in feed and water consumption with loss in body condition together with greenish-yellow diarrhoea was generally noted. Peritonitis and pericarditis were the most common findings in young budgerigars at necropsy, while in adult budgerigars scattered grey-white necrotic foci were found in the livers. Histopathologically, the lesions in young budgerigars were characterized with fibrinonecrotic peritonitis and/or pericarditis and necrotic hepatitis. In adult budgerigars with acute infection, hepatic necrosis with focal heterophil infiltration was present; whilst lesions in the chronic cases were granulomatous in nature with the infiltration of macrophages, lymphocytes and histiocytes. For the detection of S. Gallinarum in formalin-fixed, paraffin-embedded tissues, the avidin-biotin peroxidase complex and immunofluorescence methods were used. Both methods showed bacteria to be localized in the liver, kidney, peritoneum, heart, spleen and intestines of both young and adult budgerigars. The results of the present study indicate that the avidin-biotin peroxidase complex method was more sensitive than the immunofluorescence method in the detection of the bacteria.

Tetrathyridiosis and tubulopapillary carcinoma occurring simultaneously in the mammary gland of a cat.

This report describes a case of tubulopapillary carcinoma and concomitant tetrathyridiosis in a 5-year-old female cross-breed cat. A mass was located at right inguinal mammary gland and measured 5.5 × 5 × 3 cm in size with multilobulated to solid appearance. The cut surface of the mass had a centrally located large cyst (approximately 3 cm in diameter) surrounded by other smaller cysts. Histologically, the mass was diagnosed as tubulopapillary mammary carcinoma, intensely positive for AE1/AE3 cytokeratins. The cyst found at post-mortem examination was tetrathyridia of Mesocestoides species surrounded by inflammatory cells and a loose fibrous capsule. To the authors' best knowledge, this is the first description of a tubulopapillary carcinoma and tetrathyridiosis found simultaneously in the mammary gland of a cat.

Relationship between anosmia and hospitalisation in patients with coronavirus disease 2019: an otolaryngological perspective.

OBJECTIVE: A study was carried out to evaluate the relationship between anosmia and hospital admission in coronavirus disease 2019 patients. METHODS: The clinical data of 1534 patients with confirmed coronavirus disease 2019 virus were analysed. The study was conducted with medical records of 1197 patients (78 per cent). The basic characteristics of patients and symptoms related to otolaryngology practice were examined. The patients were divided into two groups according to their follow up: an out-patient group and an in-patient group. RESULTS: The majority of patients presented with anosmia (44.2 per cent), dysgeusia (43.9 per cent) and fever (38.7 per cent). Anosmia was observed in 462 patients (47 per cent) in the out-patient group, and in only 67 patients (31.2 per cent) in the in-patient group. Younger age (odds ratio = 1.05, 95 per cent confidence interval = 1.03-1.06) and the presence of anosmia (odds ratio = 2.04, 95 per cent confidence interval = 1.39-3) were significantly related to out-patient treatment. CONCLUSION: Anosmia could be a symptom in the clinical presentation of the coronavirus disease 2019 infection.

A comparative study of the prevalence of the metabolic syndrome and its components in type 2 diabetic patients in two Caribbean islands using the new International Diabetes Federation definition.

BACKGROUND AND AIM: Tobago and Trinidad are two Caribbean islands with distinct genetic background and lifestyles; while Tobago is serene and a tourist centre, Trinidad is characterized by a hustling and bustling lifestyle. The study was aimed at determining and comparing the prevalence of the metabolic syndrome (MetS) and its critical components in type 2 diabetic patients using the new International Diabetes Federation (IDF) definition. METHODS: Four hundred and thirteen (166 Tobago, 247 Trinidad) type 2 diabetic patients visiting 10 lifestyle disease clinics were studied. Blood pressure, anthropometric parameters (height, weight, body mass index and waist circumference) and overnight fasting blood samples were taken. Plasma glucose and serum triglycerides, total cholesterol, LDL- and HDL-cholesterol, insulin, and adiponectin were determined. Insulin resistance (IR) was determined using the HOMA method. RESULTS: The patients in Tobago were significantly older than patients in Trinidad (p < 0.001) but the duration of diabetes (9.4 +/- 0.5 vs. 11.1 +/- 0.7 yr), medications, generalized (31.7 vs. 38.8%) and central (78.5 vs. 83.7%) obesity were similar (p > 0.05). In comparison with patients in Tobago, diabetic patients in Trinidad, irrespective of gender, had significantly higher prevalence of IDF critical components such as raised BP, raised triglycerides and reduced HDL-cholesterol (all, p < 0.001). Thus, while more patients in Trinidad were diagnosed with MetS based on three or four components, more patients in Tobago were diagnosed based on two components (p < 0.001). CONCLUSIONS: There were high prevalence rates of the components of the MetS in both the islands of Tobago and Trinidad. Quantitatively, the aggregation of the components is higher in patients in Trinidad, which constitute greater risk for adverse cardiovascular outcome. Controlling central obesity should be the target in preventing MetS in the two islands.

Protective effects of silymarin and curcumin on cyclophosphamide-induced cardiotoxicity.

INTRODUCTION: Cyclophosphamide (CP) is a potent anticancer agent; its clinical use is limited due to its marked cardiotoxicity. AIM: The present study was aimed at evaluating the cardioprotective effects of silymarin (SLY) and curcumin (CUR), which have strong antioxidant properties, against the toxic effects of high-dose CP on the heart of rats. MATERIALS AND METHODS: A total of 36 adult Wistar albino female rats were randomly divided into six groups. Group I (control group; nothing was administered), Group II (CP group; 30mg/kg/day CP was administered intraperitoneally to each animal for seven days), Group III (SLY group; 100mg/kg/day SLY by gavage for 14 days), Group IV (CUR group; 100mg/kg/day CUR by gavage for 14 days), Group V (SLY+CP group; 100mg/kg/day SLY by gavage for 14days plus 30mg/kg/day CP intraperitoneally starting from the seventh day) and Group VI (CUR+CP group; 100mg/kg/day CUR by gavage for 14days plus 30mg/kg/day CP intraperitoneally starting from the seventh day). Biochemical, histopathological and immunohistochemical methods were utilised for evaluation of the cardiotoxicity. RESULTS: The result showed that an increase in heart MDA and DNA fragmentation levels were detected while significant decreases were seen in SOD levels in CP alone group when compared to the other groups. CP caused severe damage in the histopathological status of heart tissue including intersititial oedema, haemorrhage, degeneration and necrosis in muscle fibrils and perinuclear vacuolization. A significant increase in the percentage of TUNEL-positive cells and γH2AX protein expression was detected in the CP-treated group compared to the control and other treated groups. There was significant increase in the percentage of caspase 3-positive cells and decrease in the percentage of Bcl-2 positive cells in the CP group compared to the control group and other treated groups. However, a significant decrease in the percentage of cTnI and cTnT immunoreactivity was also observed in the CP-treated group compared to the control and other treated groups. In the groups in which SLY and CUR were administered concurrently with CP, biochemical parameters, histopathological and immunohistochemical results were found to be significantly lower than in the CP-only group. CONCLUSIONS: These results lead to conclusion that the natural antioxidant SLY and CUR might have protective effects against CP-induced cardiotoxicity and oxidative stress in rats.

Altered neurite morphology and cholinergic function of induced pluripotent stem cell-derived neurons from a patient with Kleefstra syndrome and autism.

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD.

Medicolegal aspects of rhinology practice.

OBJECTIVE: To investigate rhinology-related malpractice claims with the aim of optimising safe practice. METHODS: The database of the National Institute of Forensic Medicine was reviewed. In total, 241 otorhinolaryngology malpractice case reports dating from 2005 to 2012 were evaluated, and 83 malpractice cases related to rhinology treatments were separated. RESULTS: There was no significant difference between the number of male (n = 42) and female (n = 41) claimants. The mean patient age was 32.07 ± 10.53 years (range, 10-75 years). Seventy-nine cases involved surgical treatment in rhinology. The most common complaints were: unsatisfactory cosmetic results (n = 30), optic nerve injury (n = 10), septal perforation (n = 9) and intracranial penetration (n = 4). Malpractice was detected in 21 cases (25.3 per cent). No delinquency was found in 62 cases (74.7 per cent). CONCLUSION: Physicians should be aware of legal consequences related to rhinology practice. Further study is needed on this topic, as well as interdisciplinary collaboration, to ensure best practices and to avoid litigation.

Reduced phosphorylation of transcription factor Elk-1 in cultured fibroblasts of a patient with premature aging syndrome and insulin resistance.

The effect of insulin and growth factor mediated signaling to gene regulation was investigated in cultured fibroblasts of a patient with a premature aging syndrome (metageria) and severe insulin resistance. Insulin receptor structure and function as well as major pathways activated by insulin, i.e. phosphatidyl inositol-3 kinase (PI-3 K) cascade or mitogen-activated protein kinase (MAPK) cascades, were functional. Inducibility of the proto-oncogene cfos, a representative endpoint of signaling pathways related to gene expression, by growth factors or insulin was reduced in patient cells. This reduced induction persisted in cfos promoter reporter gene studies indicating that the post receptor defect is localized proximal to the cfos promoter itself. Abundances of the transcription factors Elk-1 and SRF being major players in coupling of MAPKs to cfos promoter activation were not altered. However, basal and inducible phosphorylation of Elk-1 was impaired. In addition, basal and stimulated transcriptional activity mediated by Elk-1 was almost abolished in patient cells. Therefore these results identify a post receptor defect in cFos induction, which appears to be related to a functional alteration of Elk-1. A possible relation of this signal transduction defect to the specific premature aging syndrome remains to be elucidated.

Preparation of antibacterial PVA and PEO nanofibers containing Lawsonia Inermis (henna) leaf extracts.

Concerns about health issues and environmental pollution stimulate research to find new health and hygiene related products with healing properties and minimum negative effect on the environment. Development of new, natural antibacterial agents has become one of the most important research areas to combat some pathogens such as Gram- positive and Gram-negative bacteria, fungi, algae, yeast, and some microorganisms which cause serious human infections. Lawsonia Inermis (henna) leaf extracts for preparation of antibacterial poly(ethylene oxide) (PEO) and poly(vinyl alcohol) (PVA) nanofibers via electrospinning technique were investigated. PEO and PVA based electrospun fibers containing henna extract were verified by the appearance of FTIR peaks corresponding to the pure extract. Our study demonstrates that 2.793 wt.% Li in PVA and PEO based solutions showed bactericidal effects against Staphylococcus aureus and bacteriostatic action to Escherichia coli. Concentrations of henna leaf extract strongly impacted antibacterial activities against both bacteria. Henna leaves have a great potential to be used as a source of a potent eco-friendly antimicrobial agent.

Properties of antibacterial polypropylene/nanometal composite fibers.

Melt spinning of polypropylene fibers containing silver and zinc nanoparticles was investigated. The nanometals were generally uniformly dispersed in polypropylene, but aggregation of these materials was observed on fiber surface and in fiber cross-sections. The mechanical properties of the resulted composite fibers with low concentration of nanometal were comparable to those for the control PP yarns. Extruded composite fibers that contained 0.72% silver and 0.60% zinc nanoparticles had outstanding antibacterial efficacy as documented by the percentage count reduction growth of Escherichia coli and Staphylococcus aureus. Fibers containing silver particles had improved antistatic properties.

Prediction of 10-year coronary heart disease risk in Caribbean type 2 diabetic patients using the UKPDS risk engine.

OBJECTIVE: Primary prevention of Coronary Heart Disease (CHD) in diabetic patients should be based on absolute CHD risk calculation. This study was aimed to determine the levels of 10-year CHD risk in Caribbean type 2 diabetic patients using the diabetes specific United Kingdom Prospective Diabetes Study (UKPDS) risk engine calculator. SUBJECTS AND METHODS: Three hundred and twenty-five (106 males, 219 females) type 2 diabetic patients resident in two Caribbean Islands of Tobago and Trinidad met the UKPDS risk engine inclusion criteria. Records of their sex, age, ethnicity, smoking habit, diabetes duration, systolic blood pressure, total cholesterol, HDL-cholesterol and glycated haemoglobin were entered into the UKPDS risk engine calculator programme and the absolute 10-year CHD and stroke risk levels were computed. The 10-year CHD and stroke risks were statistically stratified into <15%, 15-30% and >30% CHD risk levels and differences between patients of African and Asian-Indian origin were compared. RESULTS: In comparison with patients in Tobago, type 2 diabetic patients in Trinidad, irrespective of gender, had higher proportion of 10-year CHD risk (10.4 vs. 23.6%, P<0.001) whereas the overall 10-year stroke risk prediction was higher in patients resident in Tobago (16.9 vs. 11.4%, P<0.001). Ethnicity-based analysis revealed that irrespective of gender, higher proportion of patients of Indian origin scored >30% of absolute 10-year CHD risk compared with patients of African descent (3.2 vs. 28.2%, P<0.001). CONCLUSIONS: The results of the study identified diabetic patients resident in Trinidad and patients of Indian origin as the most vulnerable groups for CHD. These groups of diabetic patients should have priority in primary or secondary prevention of coronary heart disease.

Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons.

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.

Insulin-regulated transcription factors: molecular link between insulin resistance and cardiovascular risk factors.

Patients with insulin resistance and/or type 2 diabetes have a 5-fold increase in cardiovascular mortality rate. Therefore, it is a current issue of discussion that arterial hypertension, lipid disorders as well as visceral obesity are coronary risk factors, which might belong to a syndrome that is caused by decreased insulin sensitivity. Concerning a possible molecular link between insulin resistance, atherosclerosis and obesity, we focus in our research on questions looking for a molecular link between lipid metabolism, insulin action, and obesity at a gene regulatory level. Alterations in the structure, function and regulation of transcription factors appear to be such signalling steps which might play an essential role in the pathogenesis and therapy of cardiovascular risk factors associated with insulin resistance, eg the so called metabolic syndrome. Recent examples are members of the nuclear hormone receptor superfamily, eg peroxisome proliferator-activated receptor (PPAR) isoforms and sterol regulatory element-binding proteins (SREBPs). Beside their regulation by different metabolites, these transcription factors are also targets of hormones, like insulin and leptin, growth factors, and inflammatory signals. Therefore, they appear to be a point of signalling convergence at a gene regulatory level. Major signalling pathways coupling receptors at the cell surface for hormones, growth factors as well as cytokines to gene regulatory events in the nucleus are the MAP-kinase cascades. We have recently defined different postreceptor defects in these pathways in patients with clinical phenotypes corresponding to congenital lipoatrophy. Therefore, these studies may identify novel pathways which play a role in the control of body weight, insulin sensitivity and cardiovascular risk.

A single nucleotide substitution in the promoter region of the apolipoprotein C-II gene identified in individuals with chylomicronemia.

Apolipoprotein (apo) C-II plays a major role as a cofactor for lipoprotein lipase, the enzyme involved in the hydrolysis of triglyceride-rich particles. We identified in two relatives of a family (mother and son) massive hypertriglyceridemia with chylomicronemia. In these individuals apoC-II was not measurable in plasma by radial immunodiffusion. On isoelectric focusing of very low density apolipoproteins, trace amounts of apoC-II became obvious in the regular position. By sequencing, no abnormalities in the exons or neighboring intron sequences were detected. However, three alterations in the DNA sequence were found upstream from the transcription initiation site. Two variations could be explained by differences in previously published DNA sequences. The third variation (A-->G; position -86; Das et al. 1987. J. Biol. Chem. 262: 4787-4793) was present only in the homozygous form in the two hypertriglyceridemic probands. In 46 hypertriglyceridemic individuals outside the family, this mutation was not found. In electrophoretic mobility shift experiments with nuclear extracts from HepG2 cells, the 31 bp DNA fragment carrying the A-->G substitution resulted in a markedly diminished protein binding compared with the wildtype DNA fragment. In promoter reporter gene assays, the activity of the basal promoter was reduced in the case of the A-->G substitution and the deletion of the bases -91 to -58. The pedigree analysis and the experimental results are evidence that this is the first mutation in the apolipoprotein C-II gene where a single nucleotide substitution diminishes the binding of a transcription factor to a positive cis-acting clement in the promoter resulting in a depletion of apolipoprotein C-II in plasma.

Characterization of a postreceptor signaling defect that impairs cfos expression in cultured fibroblasts of a patient with insulin resistance.

Induction of cfos expression is a definite end point of signal transduction by receptor tyrosine kinases via MAPK cascades. We have examined signal transduction to transcription factor cFos in isolated fibroblasts of a patient with an inherited syndrome of insulin resistance. MAPK phosphorylation and activity were unaltered, but inducibility of cfos transcription was strongly impaired by insulin and reduced by PDGF. Induction of the cfos promoter via MAPK is mediated by activation of the ternary complex. Abundance of SRF or Elk-1 was unaltered, but Elk-1 phosphorylation following stimulation was reduced. Transient transfections with reporter genes under control of the Elk-1 binding ets/sre cis element or expression plasmids coding for the regulatory domain of Elk-1 fused to heterologous DNA binding domains revealed a defect of Elk-1 activation in the patient cells. These data identify a novel postreceptor defect of insulin and growth factors involving activation of transcription.

SREBP-1 mediates activation of the low density lipoprotein receptor promoter by insulin and insulin-like growth factor-I.

Transcription of the low density lipoprotein (LDL) receptor gene is regulated by intracellular cholesterol concentration, hormones, and growth factors. We studied the mechanisms by which insulin and estradiol stimulate promoter activity of the LDL receptor gene. Hormonal effects were analyzed in HepG2 cells after transient transfection with promotor reporter gene constructs. Successive 5' deletions of the LDL receptor promoter fragment from -537 to +88 revealed the sterol regulatory element 1 (SRE-1) between -65 and -56 as an insulin- and estradiol-sensitive cis-element. If the SRE-1 is point mutated at position -59 (C to G), which abolishes the binding of the SRE binding proteins (SREBP-1 and SREBP-2), no insulin or estradiol stimulatory effect on reporter gene expression was observed, indicating a role of SRE binding proteins in this regulatory mechanism. The concentration of the 125-kDa membrane-integrated SREBP-1 precursor protein in LDL repressed HepG2 cells is not altered by hormone treatment. Concentrations of SREBP-1 mRNA and precursor protein are reduced significantly by high and stable expression of an SREBP-1 antisense cDNA fragment in HepG2 cells (SREBP1(-) cells). Transfection of SREBP1(-) cells with promoter construct phLDL4 (-105 to +88) reduces induction of reporter gene activity by insulin and insulin-like growth factor-I to 35 and 17%, respectively, compared with HepG2 cells. The stimulatory effect of estradiol remains unchanged, and the inductions by pravastatin are enlarged. We conclude that different regulatory effects converge at SRE-1, but that SREBP-1 is selectively involved in the signal transduction pathway of insulin and insulin-like growth factor-I leading to LDL receptor gene activation.

Structure of the human apolipoprotein C-II gene promoter.

To localize transcriptional active cis elements and to study the effect of a single base transition at -86 derived from a patient (PS) with an inherited severe form of hypertriglyceridemia a reporter gene transfection assay with apo C-II promoter fragments was performed. Sequences from -170 to -140, -140 to -59 and -59 to -39 were transcriptional active. The A to G transition at -86 reduced promoter activity to 25% (pC2PS-13). A nuclear protein bound sequence specific to a DNA fragment from -102 to -70, but disappeared when the point mutation at -86 was introduced. We found that one of these cis elements enhances promoter activity as a response of cAMP elevation. Apo CII mRNA level increases after incubation of HepG2 cells with 10 microM forskolin within 2 hours as measured by northern blot analyses. Incubation of transfected cells (pC2W) with forskolin [10 microM] or a cAMP analogue resulted in an 6-fold increase of luciferase activity within 6 hours and a subsequent decrease. The results suggest that several positive acting elements are located between -170 and -36 and that one of them may be responsible for promoter activation by cAMP. The reduction of promoter activity by the point mutation may be a consequence of impaired protein-DNA interaction.