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1.
G3 (Bethesda) ; 14(4)2024 04 03.
Article in English | MEDLINE | ID: mdl-38366577

ABSTRACT

High-throughput sequencing-based methods for bulked segregant analysis (BSA) allow for the rapid identification of genetic markers associated with traits of interest. BSA studies have successfully identified qualitative (binary) and quantitative trait loci (QTLs) using QTL mapping. However, most require population structures that fit the models available and a reference genome. Instead, high-throughput short-read sequencing can be combined with BSA of k-mers (BSA-k-mer) to map traits that appear refractory to standard approaches. This method can be applied to any organism and is particularly useful for species with genomes diverged from the closest sequenced genome. It is also instrumental when dealing with highly heterozygous and potentially polyploid genomes without phased haplotype assemblies and for which a single haplotype can control a trait. Finally, it is flexible in terms of population structure. Here, we apply the BSA-k-mer method for the rapid identification of candidate regions related to seed spot and seed size in diploid potato. Using a mixture of F1 and F2 individuals from a cross between 2 highly heterozygous parents, candidate sequences were identified for each trait using the BSA-k-mer approach. Using parental reads, we were able to determine the parental origin of the loci. Finally, we mapped the identified k-mers to a closely related potato genome to validate the method and determine the genomic loci underlying these sequences. The location identified for the seed spot matches with previously identified loci associated with pigmentation in potato. The loci associated with seed size are novel. Both loci are relevant in future breeding toward true seeds in potato.


Subject(s)
Solanum tuberosum , Humans , Solanum tuberosum/genetics , Plant Breeding , Chromosome Mapping/methods , Quantitative Trait Loci , Seeds/genetics
2.
Toxicol Mech Methods ; 27(6): 451-457, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28413915

ABSTRACT

Patients with hypogonadism are at increased risk of cardiac and metabolic diseases. However, the pathogenesis of increased cardiometabolic risk in patients with hypogonadism is not clear. Oxidative stress plays an important role in the pathogenesis of cardiometabolic diseases. This study aimed to investigate possible differences in oxidative stress conditions between patients with hypogonadism and healthy controls. In this study, 38 male patients with congenital hypogonadotropic hypogonadism (CHH) (mean age: 21.7 ± 1.6 years) and 44 healthy male controls (mean age: 22.3 ± 1.4 years) with almost equal body mass index were enrolled. The demographic parameters, follicle-stimulating hormone (FSH), luteinizing hormone (LH), total and free testosterone, homeostatic model assessment of insulin resistance (HOMA-IR) and oxidative stress parameters, such as superoxide dismutase, catalase (CAT), glutathione peroxidase (GPx) and malondialdehyde (MDA), were compared between both groups. Compared to the healthy controls, triglycerides (p = .02), insulin levels, HOMA-IR values, CAT activities and MDA levels (p < .001 for all) were significantly higher and HDL cholesterol (p = .04), total and free testosterone, FSH, LH levels and GPx activity were significantly lower (p < .001 for all) in patients with CHH. There were significant correlations between total testosterone levels and CAT activity (r = -.33 p = .01), GPx activity (r = .36 p = .007) and MDA (r = -.47 p < .001) levels. The results of this study showed that young and treatment-naïve patients with congenital hypogonadism had an increased status of oxidative stress.


Subject(s)
Catalase/blood , Glutathione Peroxidase/blood , Hypogonadism/blood , Malondialdehyde/blood , Oxidative Stress , Testosterone/blood , Biomarkers/blood , Case-Control Studies , Erythrocytes/enzymology , Follicle Stimulating Hormone/blood , Humans , Hypogonadism/congenital , Lipids/blood , Luteinizing Hormone/blood , Male , Superoxide Dismutase/blood , Young Adult
3.
Rev. esp. med. nucl. imagen mol. (Ed. impr.) ; 35(5): 321-324, sept.-oct. 2016. ilus
Article in English | IBECS | ID: ibc-155016

ABSTRACT

We present a patient with a complex clinical picture of primary hyperparathyroidism with multiple destructive skeletal lesions suspicious of bone metastases and concomitant multifocal papillary thyroid carcinoma with a metastatic central lymph node. He presented with progressively worsening right hip pain and restricted motion. Magnetic resonance imaging revealed multiple lytic lesions involving predominantly the right trochanter minor and the left inferior and posterior pubic rami. Biochemical tests were consistent with primary hyperparathyroidism. Neck ultrasound and parathyroid scintigraphy revealed a single parathyroid adenoma and a thyroid nodule, preoperative cytology of which confirmed papillary thyroid carcinoma, as did the final surgical specimen. Biochemical results, regarding hyperparathyroidism, declined to normal levels and his complaints gradually decreased after surgery. Postoperative whole body bone scintigraphy showed increased tracer uptakes at multiple sites, but they were proved to be metabolically inactive by fluorodeoxyglucose positron emission tomography/computed tomography (AU)


Presentamos el caso de un paciente con un cuadro clínico de hiperparatiroidismo primario, con muchas lesiones óseas destructivas sospechosas de metástasis óseas y carcinoma tiroideo multifocal concomitante con un ganglio linfático metastásico central. Se presentó con agravamiento progresivo de dolor y restricción de movimiento en la cadera derecha. La resonancia magnética reveló múltiples lesiones líticas que implicaban principalmente al trocánter menor derecho y a las ramas púbicas izquierdas inferior y posterior. Las pruebas bioquímicas fueron consistentes con un hiperparatiroidismo primario. La ecografía cervical y la gammagrafía paratiroidea revelaron un único adenoma paratiroideo y un nódulo tiroideo, cuya citología preoperatoria confirmó un carcinoma papilar de tiroides, que fue confirmado también por la muestra final obtenida quirúrgicamente. Los resultados bioquímicos, en relación con el hiperparatiroidismo, descendieron a niveles normales tras la cirugía, y los dolores fueron remitiendo gradualmente. La gammagrafía ósea postoperatoria de cuerpo entero reflejó un incremento de captación del radiotrazador en múltiples localizaciones, que resultaron ser metabólicamente inactivas en la tomografía por emisión de positrones con fluorodesoxiglucosa/tomografía computarizada (AU)


Subject(s)
Humans , Male , Adult , Parathyroid Neoplasms/complications , Parathyroid Neoplasms/surgery , Parathyroid Neoplasms , Radionuclide Imaging/methods , Hyperparathyroidism/complications , Hyperparathyroidism , Carcinoma/surgery , Carcinoma , Technetium/analysis , Technetium Tc 99m Sestamibi/analysis , Positron-Emission Tomography/methods , Positron-Emission Tomography , Radioactive Tracers , Clinical Chemistry Tests
4.
Rev Esp Med Nucl Imagen Mol ; 35(5): 321-4, 2016.
Article in English, Spanish | MEDLINE | ID: mdl-27036887

ABSTRACT

We present a patient with a complex clinical picture of primary hyperparathyroidism with multiple destructive skeletal lesions suspicious of bone metastases and concomitant multifocal papillary thyroid carcinoma with a metastatic central lymph node. He presented with progressively worsening right hip pain and restricted motion. Magnetic resonance imaging revealed multiple lytic lesions involving predominantly the right trochanter minor and the left inferior and posterior pubic rami. Biochemical tests were consistent with primary hyperparathyroidism. Neck ultrasound and parathyroid scintigraphy revealed a single parathyroid adenoma and a thyroid nodule, preoperative cytology of which confirmed papillary thyroid carcinoma, as did the final surgical specimen. Biochemical results, regarding hyperparathyroidism, declined to normal levels and his complaints gradually decreased after surgery. Postoperative whole body bone scintigraphy showed increased tracer uptakes at multiple sites, but they were proved to be metabolically inactive by fluorodeoxyglucose positron emission tomography/computed tomography.


Subject(s)
Carcinoma, Papillary/complications , Hyperparathyroidism, Primary/complications , Hyperparathyroidism, Primary/pathology , Thyroid Neoplasms/complications , Adult , Humans , Male , Thyroid Cancer, Papillary
5.
Curr Protoc Plant Biol ; 1(2): 431-450, 2016 Mar.
Article in English | MEDLINE | ID: mdl-30775870

ABSTRACT

Establishing efficient functional genomic systems for creating and characterizing genetic variation in forest trees is challenging. Here we describe protocols for creating novel gene-dosage variation in Populus through gamma-irradiation of pollen, followed by genomic analysis to identify chromosomal regions that have been deleted or inserted in each F1 individual. Irradiated pollen is used in a controlled, interspecific cross to create F1 progeny that carry deletions and insertions of chromosomal regions. These insertions and deletions result in novel changes in gene dosage that in turn affect both qualitative and quantitative phenotypic traits. The protocols described here outline the processes involved in optimizing irradiation levels and performing controlled crosses, sowing seed and propagating seedlings, and genome-wide sequence-based analysis of deletions and insertions in the F1 progeny. The same approach could be applied to other vegetatively propagated species. © 2016 by John Wiley & Sons, Inc.

6.
PLoS One ; 10(10): e0139672, 2015.
Article in English | MEDLINE | ID: mdl-26445462

ABSTRACT

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.


Subject(s)
Agrobacterium tumefaciens/genetics , Arabidopsis/genetics , DNA, Bacterial/analysis , DNA, Bacterial/genetics , High-Throughput Nucleotide Sequencing/methods , Plants, Genetically Modified/genetics , Transformation, Genetic , DNA, Plant/genetics , Genome, Plant , High-Throughput Nucleotide Sequencing/economics , Mutagenesis, Insertional , Sequence Analysis, DNA/economics , Sequence Analysis, DNA/methods
7.
Horm Metab Res ; 46(13): 955-8, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25181418

ABSTRACT

Cardiometabolic disorders and osteoporosis are prevalent in patients with hypogonadism. Osteoprotegerin (OPG) and fibroblast growth factor-23 (FGF-23), are co-secreted from bones and vascular endothelium, regulating bone mineral metabolism and vascular functions. Vitamin D is another hormone with dual effects on bone and vascular metabolism. The aim of this study was to search for any difference between the serum levels of OPG, FGF-23, and vitamin D in patients with hypogonadism and the healthy controls. We also aimed to search for any relationship between these parameters and endothelial dysfunction or insulin resistance. Forty-nine male patients with congenital hypogonadotropic hypogonadism (CHH) (mean age 20.71 ± 1.75 years) and 43 BMI matched healthy male subjects (mean age 21.37 ± 1.04 years) were enrolled. OPG, FGF-23, vitamin D, and asymmetric dimethylarginine (ADMA) levels were measured from the fasting serum samples. The insulin sensitivity was estimated by homeostatic model assessment-insulin resistance (HOMA-IR) formula. Triglycerides, insulin, HOMA-IR, and ADMA levels in the patient group were significantly higher than the values of the control group (p = 0.014, p = 0.002, p = 0.003, p < 0.001, respectively). The OPG, FGF-23, and vitamin D levels of the patients were not significantly different from the healthy controls. In addition, these markers were not correlated to ADMA or HOMA-IR levels. The results show that young and treatment naive subjects with CHH have endothelial dysfunction and insulin resistance when compared to their healthy counterparts. However, the OPG, FGF-23, and vitamin D levels were similar in the 2 groups. In addition, these parameters are not significantly related to the endothelial functions or insulin resistance in these subjects.


Subject(s)
Cholecalciferol/blood , Fibroblast Growth Factors/blood , Hypogonadism/blood , Osteoprotegerin/blood , Case-Control Studies , Demography , Fibroblast Growth Factor-23 , Humans , Male , Young Adult
8.
Plant Cell ; 26(4): 1382-1397, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24728647

ABSTRACT

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but systematic cataloguing of mutations would further increase their utility. We examined the suitability of multiplexed global exome capture and sequencing coupled with custom-developed bioinformatics tools to identify mutations in well-characterized mutant populations of rice (Oryza sativa) and wheat (Triticum aestivum). In rice, we identified ∼18,000 induced mutations from 72 independent M2 individuals. Functional evaluation indicated the recovery of potentially deleterious mutations for >2600 genes. We further observed that specific sequence and cytosine methylation patterns surrounding the targeted guanine residues strongly affect their probability to be alkylated by ethyl methanesulfonate. Application of these methods to six independent M2 lines of tetraploid wheat demonstrated that our bioinformatics pipeline is applicable to polyploids. In conclusion, we provide a method for developing large-scale induced mutation resources with relatively small investments that is applicable to resource-poor organisms. Furthermore, our results demonstrate that large libraries of sequenced mutations can be readily generated, providing enhanced opportunities to study gene function and assess the effect of sequence and chromatin context on mutations.

9.
Horm Metab Res ; 45(6): 443-8, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23613012

ABSTRACT

Metabolic disorders and cardiovascular events are increased in hypogonadism. Serum HDL composition is a better cardiovascular predictor than the HDL counts. However, there is no information about the HDL subfractions in patients with hypogonadism. We designed a prospective study to investigate the HDL subfractions in treatment naïve subjects with hypogonadism and the effects of 2 different testosterone replacement regimens on the HDL subfractions. Seventy young male patients with congenital hypogonadotropic hypogonadism (CHH) and 70 age and BMI-matched healthy males were enrolled in the present study. The patients were assigned to receive intramuscular injections of testosterone esters 250 mg every 3 weeks and transdermal testosterone applications 50 mg daily. Biochemical investigations including HDL subfractions and insulin resistance were done. Patients with CHH had higher levels of insulin, HOMA-IR, WC, triglyceride, and diastolic blood pressure. Although, the HDL cholesterol concentrations were similar in both groups, hypogonadal patients had lower HDL2 and higher HDL3 levels. The total testosterone levels were independent determinants of the HDL2 subfractions. During the follow-up, a significant increase in the BMI and WC values and a significant decrease in the levels of total cholesterol, HDL cholesterol, and HDL3 were observed. No difference was present between the 2 treatment arms. These results show that patients with hypogonadism have unfavorable HDL compositions in addition to the other dysmetabolic features. However, testosterone replacement for about six months neither improves the metabolic problems nor the HDL composition. Mechanistic studies are warranted to better understand the cardiovascular effects of unfavorable HDL compositions in hypogonadism.


Subject(s)
Cholesterol, HDL/metabolism , Hormone Replacement Therapy , Hypogonadism/drug therapy , Testosterone/administration & dosage , Case-Control Studies , Cholesterol, HDL/analysis , Humans , Hypogonadism/congenital , Hypogonadism/metabolism , Lipoproteins, HDL/analysis , Lipoproteins, HDL/metabolism , Male , Prospective Studies , Young Adult
10.
Exp Clin Endocrinol Diabetes ; 120(5): 261-5, 2012 May.
Article in English | MEDLINE | ID: mdl-22549343

ABSTRACT

Polycystic ovary syndrome (PCOS) is characterized by insulin resistance. Chronic low grade inflammation has been reported to participate in the pathogenesis of insulin resistance. Chitotriosidase (ChT), a protein secreted by activated macrophages, has been shown to be involved in chronic inflammatory responses. In the present study, serum chitotriosidase activity and its relationship with insulin resistance were determined in patients with PCOS.34 patients with PCOS and 44 age and body mass index (BMI) matched healthy controls were enrolled in the study. ChT activity was measured by the fluorescence method. High sensitivity C reactive protein (hs-CRP) and adiponectin levels were determined by enzyme immunoassay (EIA). Insulin resistance was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR) formula.Plasma ChT activity, hs-CRP level and HOMA-IR score were significantly higher (p=0.024, p=0.002, p=0.001, respectively) while plasma adiponectin concentration was significantly lower (p=0.018) in women with PCOS compared to healthy controls. Blood ChT activity correlated positively with age, waist-to-hip ratio (WHR), BMI, hs-CRP, HOMA-IR and negatively with blood adiponectin level. After adjustment for age and BMI, ChT activity, total testosterone level and WHR remained as the independent predictors of HOMA-IR score in logistic regression analysis.ChT activity is increased in patients with PCOS in concordance with insulin resistance. These findings may reflect the pronounced risk for metabolic syndrome and atherosclerotic diseases in this particular patient group.


Subject(s)
Hexosaminidases/blood , Inflammation/enzymology , Insulin Resistance , Polycystic Ovary Syndrome/enzymology , Adiponectin/blood , C-Reactive Protein/metabolism , Female , Humans , Inflammation/blood , Linear Models , Polycystic Ovary Syndrome/blood , Waist-Hip Ratio , Young Adult
11.
Eur J Endocrinol ; 164(5): 759-64, 2011 May.
Article in English | MEDLINE | ID: mdl-21325471

ABSTRACT

OBJECTIVE: The relationship between metabolic syndrome (MS) and hypogonadism has always been investigated in study groups confounded with aging, obesity or chronic metabolic disorders. So far, there has been no data about the presence of MS in young hypogonadal patients. Also, there is controversial data about the metabolic effects of testosterone replacement therapy. We investigated the frequency of MS in treatment-naïve, young men with congenital hypogonadal hypogonadism (CHH). We also searched for the effect of testosterone replacement on the metabolic profiles of this specific patient group. DESIGN: Retrospective analysis. METHODS: A total of 332 patients (age 21.68 ± 2.09 years) were enrolled. The control group included 395 age- and body mass index (BMI)-matched healthy young men (age 21.39 ± 1.49 years). Standard regimen of testosterone esters (250 mg/3 weeks) was given to 208 patients. RESULTS: MS was more prevalent in CHH (P<0.001) according to healthy controls. The patients had higher arterial blood pressure, waist circumference (WC), triglyceride (P<0.001 for all), fasting glucose (P=0.02), fasting insulin (P=0.004), homeostatic model assessment of insulin resistance (HOMA-IR) (P=0.002) and lower high density lipoprotein (HDL) cholesterol (P<0.001) levels. After 5.63±2.6 months of testosterone treatment, the BMI, WC (P<0.001 for both), systolic blood pressure (P=0.002) and triglyceride level (P=0.04) were increased and the total and HDL cholesterol levels were decreased (P=0.02 and P<0.001 respectively). CONCLUSIONS: This study shows increased prevalence of MS and unfavorable effects of testosterone replacement in young patients with CHH. Long-term follow-up studies are warranted to investigate the cardiovascular safety of testosterone treatment in this specific population.


Subject(s)
Hormone Replacement Therapy/adverse effects , Hypogonadism/blood , Hypogonadism/drug therapy , Metabolic Syndrome/blood , Metabolic Syndrome/chemically induced , Testosterone/adverse effects , Follow-Up Studies , Humans , Male , Metabolic Syndrome/epidemiology , Retrospective Studies , Testosterone/therapeutic use , Young Adult
12.
Handchir Mikrochir Plast Chir ; 42(4): 233-8, 2010 Aug.
Article in German | MEDLINE | ID: mdl-20235008

ABSTRACT

Femoral nerve palsy, mostly of iatrogen cause, leads to paresis of quadriceps muscle with complete loss of knee extension. Therapeutical options include neurolysis, nerve reconstruction or functional muscle transplantations. Another concept is the transfer of hamstring muscles as described in post polio surgery. We describe our experience of biceps femoris and semitendinosus muscle transfer for reconstruction of knee extension. From 2003 to 2007 seven patients (mean age 43) with complete loss of knee extension after femoral nerve lesion were treated. Nerve palsy was caused by direct lesion, traction, hematoma after collapse, lesion of lumbosacral plexus and an unclear muscle dystrophy. Indication for muscle transfer was due to long standing muscle paresis. All patients received a transfer of biceps femoris and semitendinosus muscle/tendon into the quadriceps tendon. Patients were immobilised in a cast for 6 weeks in extended knee position. Weight bearing started after 8 weeks. Operations went uneventfully. All patients were able to extend the knee postoperatively against gravity and were able to climb stairs without help. 4 Patients had complete knee extension, 2 had a lack of 20 degrees , one of 30 degrees. Daily routine was possible in all cases. No instability of knee joints occurred postoperatively. In a nerve lesion close to the muscle a nerve reconstruction should be aimed. If not performed or with unsuccessful outcome, muscle transfer is a good option to restore function. All recent studies describe good to excellent results with stable knees, allowing the patient to manage daily routine without assistance and to climb stairs up and down. Long term complications such as dislocation of patella or genu recurvatum were not observed in our patients. The latter results as typical complication in polio from weakening knee flexion through biceps femoris transfer, if the gastrocnemius muscle is not forceful enough. However in an isolated femoral nerve lesion this will rarely occur.


Subject(s)
Femoral Neuropathy/surgery , Muscle, Skeletal/transplantation , Paralysis/surgery , Quadriceps Muscle/innervation , Quadriceps Muscle/surgery , Adolescent , Adult , Aged , Child , Female , Femoral Neuropathy/etiology , Humans , Knee Joint/innervation , Knee Joint/physiopathology , Male , Middle Aged , Postoperative Care , Postoperative Complications/etiology , Postoperative Complications/surgery , Range of Motion, Articular/physiology , Reoperation , Tendon Transfer/methods
13.
Vaccine ; 27(14): 1997-2015, 2009 Mar 23.
Article in English | MEDLINE | ID: mdl-19200450

ABSTRACT

Generating broadly neutralizing antibodies with candidate vaccines has remained an elusive goal. Consequently, vaccine candidates developed have aimed at eliciting cell-mediated immune effector activities (CMI) that could delay disease progression, and maybe also limit secondary transmission, by controlling virus replication. There is considerable discussion about what types of endpoints would constitute definable standardized clinical benefit to the individual that would result in licensure of these candidate vaccines. Identifying biomarkers that can be used as surrogates for clinical endpoints in randomized clinical trials would be useful, because it would shorten studies and reduce costs. Biological markers associated with disease progression and secondary transmission and that may be used as prognosis markers and surrogate endpoints in HIV vaccine trials have emerged from analyses of data from studies on natural history of HIV infection. Extensive literature is cited to support the use of plasma viral load as a primary endpoint for supporting licensure decisions. Overall, a significant result on viral load in a vaccine trial should be considered as a significant breakthrough for vaccines and be aggressively pursued with the caveat that such a result should rapidly be followed by well-defined studies to verify durable virological and immunological vaccine benefit, as well as ultimate clinical benefit. The review also provides perspectives on magnitude of viral load reduction, durability of viral load reduction for reduced progression of HIV disease.


Subject(s)
AIDS Vaccines/immunology , HIV Infections/therapy , HIV-1/immunology , Viral Load , AIDS Vaccines/administration & dosage , Animals , Biomarkers/blood , Clinical Trials as Topic , Disease Progression , Humans , RNA, Viral/blood , T-Lymphocytes, Cytotoxic/immunology
15.
Acta Biol Hung ; 56(1-2): 129-36, 2005.
Article in English | MEDLINE | ID: mdl-15813221

ABSTRACT

In this study, chromosome number and ploidy levels of Ipheion uniflorum cv. "Wisley Blue" (spring starflower) were determined. In meristematic root tip cells, chromosome number was found as 2n = 12 and 4n = 24. The ratios of diploid and tetraploid cells were found as 80.74% and 19.26%, respectively. In differentiated root tissues and mature leaf tissues ploidy levels were analysed by flow cytometry and polysomaty were found in both organs. In differentiated root tissues, ploidy levels were found as 2C, 4C, 8C and 16C DNA. In root tissues percentages of 2C, 4C, 8C and 16C nuclear DNA content were observed as 57.2%, 33.1%, 2.47% and 7.23%, respectively. In mature leaf tissues, ploidy levels were determined 2C, 4C, 8C and 16C DNA. In this tissue the frequency of 4C DNA was found very higher (74.3%) and 2C DNA content was determined as 19.2%. In mature leaf tissue, 8C and 16C nuclear DNA contents were observed as 2.72% and 3.78%, respectively. When nuclear DNA contents in leaves and roots were compared, an apparent difference in 2C and 4C DNA contents was found.


Subject(s)
Chromosomes, Plant/ultrastructure , Magnoliopsida/physiology , Meristem/physiology , Ploidies , Cell Differentiation , Flow Cytometry , Karyotyping , Plant Leaves , Plant Physiological Phenomena , Plants
16.
Vaccine ; 19(28-29): 3972-80, 2001 Jul 16.
Article in English | MEDLINE | ID: mdl-11427273

ABSTRACT

The immune responses of mice injected with plasmids VR-gB and VR-gB Delta tm expressing the full-length membrane-anchored, or secreted forms of human cytomegalovirus (HCMV)-glycoprotein B (gB), respectively, and VR-pp65 expressing the HCMV-phosphoprotein 65 (pp65) were analyzed. Pretreatment of mice with the local anesthetic bupivacaine did not enhance antibody production, and IFN-alpha co-expressed with the immunizing plasmids induced a moderate increase in the antibody response. However, antibody response was higher in mice inoculated at three sites in the musculus quadriceps than in mice inoculated at one site with the same dose and in the same muscle. pVR-gB Delta tm induced significantly higher antibody titers than the construct expressing the membrane-anchored form of gB, and priming with pVR-gB Delta tm followed by boosting with the gB subunit resulted in high-titer antibody responses. Immunization with VR-pp65 induced dose-dependent CTL responses in about 50% of the mice at a dose of 50 microg. Co-expression of IFN-alpha did not affect the number of responding mice. These findings might be important for optimization of humoral and cellular immune responses to HCMV after DNA vaccination.


Subject(s)
Cytomegalovirus/immunology , Vaccines, DNA/pharmacology , Viral Vaccines/pharmacology , Animals , Antibodies, Viral/biosynthesis , Antigens, Viral/genetics , Bupivacaine/administration & dosage , Cytomegalovirus/genetics , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/prevention & control , Female , Humans , Immunization, Secondary , Immunoglobulin G/biosynthesis , Injections, Intramuscular , Interferon-alpha/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Phosphoproteins/genetics , Phosphoproteins/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/genetics
17.
J Infect Dis ; 183(8): 1171-9, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11262198

ABSTRACT

The major matrix phosphoprotein 65 (pp65) of cytomegalovirus (CMV) is an important target of HLA-restricted cytotoxic T cells (CTL) after natural infection. A canarypox-CMV pp65 recombinant was studied for its ability to induce CMV pp65-specific CTL, helper T lymphocytes, and antibodies in a phase I clinical trial. Twenty-one CMV-seronegative adult volunteers were randomized to receive immunizations at months 0, 1, 3, and 6 with either canarypox-CMV pp65 or placebo. In canarypox-CMV pp65-immunized subjects, pp65-specific CTL were elicited after only 2 vaccinations and were present at months 12 and 26 in all subjects tested. Cell-depletion studies indicated that the CTL were phenotype CD8(+). Peripheral blood mononuclear cells proliferated in response to stimulation with purified pp65, and antibodies specific for pp65 also were detected. Canarypox-CMV pp65 is the first recombinant vaccine to elicit CMV-specific CTL responses, which suggests the potential usefulness of this approach in preventing disease caused by CMV.


Subject(s)
Antibodies, Viral/blood , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Phosphoproteins/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, Synthetic/adverse effects , Viral Matrix Proteins/immunology , Viral Vaccines/adverse effects , Adolescent , Adult , Antibody Formation , Antibody Specificity , Avipoxvirus/genetics , Avipoxvirus/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line , Cytotoxicity, Immunologic , Genetic Vectors , Humans , Immunity, Cellular , Phosphoproteins/genetics , Viral Matrix Proteins/genetics
18.
Vaccine ; 19(13-14): 1688-93, 2001 Feb 08.
Article in English | MEDLINE | ID: mdl-11166892

ABSTRACT

Vaccination by intramuscular injection of naked DNA is very efficient in the mouse, but immunogenicity of DNA vaccines needs to be improved in man. The aim of our study was to determine in BALB/c mice if suitable electric pulses delivered to the muscle after DNA injection--a procedure called electrotransfer--could improve the immunogenicity of suboptimal doses of a DNA vaccine expressing the influenza hemagglutinin protein. The results show a significant enhancement of the cellular and antibody responses following electrotransfer for the 1- and 10-microg DNA doses, respectively, but no effect on a lower dose. At the 10-microg dose, the IgG and hemagglutination inhibition mean titres were increased 25-fold and the inter-individual variability was markedly reduced.


Subject(s)
Electric Stimulation/methods , Influenza Vaccines/administration & dosage , Influenza Vaccines/immunology , Orthomyxoviridae/immunology , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Animals , Antibodies, Viral/immunology , Cytotoxicity Tests, Immunologic , Dose-Response Relationship, Immunologic , Enzyme-Linked Immunosorbent Assay , Female , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/administration & dosage , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Immunoglobulin G/immunology , Injections, Intramuscular , Interferon-gamma/biosynthesis , Mice , Mice, Inbred BALB C , T-Lymphocytes, Cytotoxic/immunology
19.
J Infect Dis ; 181(5): 1537-46, 2000 May.
Article in English | MEDLINE | ID: mdl-10823751

ABSTRACT

The prevalence of human cytomegalovirus (HCMV) pp65-, pp150-, IE1-exon4-, gB- and pp28-specific cytotoxic T lymphocyte (CTL) responses was compared among 34 healthy individuals, grouped by neutralizing antibody titers. Moderately and highly seropositive donors showed predominantly pp65- and IE1-exon4-specific CTL responses (92% and 76% of the donors, respectively), with similar precursor frequencies in the 2 donors tested. In addition, highly seropositive and a few moderately seropositive donors showed CTL responses to gB and pp150 (33% and 30% of the donors, respectively). No individual recognized pp28 as a target in the CTL assay. Phenotypic analysis revealed a mixed effector population of CD4+ and CD8+ (1 donor) or only CD8+ cells for pp65-specific effectors (2 donors). IE1-exon4- and pp150-specific effectors were CD8+ (2 donors and 1 donor, respectively), whereas gB-specific CTLs were CD4+ (1 donor). These data may help to design a cellular immunity-based vaccine effective against HCMV diseases.


Subject(s)
Antigens, Viral/immunology , Cytomegalovirus/immunology , Immediate-Early Proteins/immunology , Phosphoproteins/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/virology , Viral Envelope Proteins/immunology , Viral Matrix Proteins/immunology , Viral Proteins/immunology , Adult , CD8-Positive T-Lymphocytes/immunology , Cytomegalovirus/genetics , Exons , Humans , Immediate-Early Proteins/genetics , Middle Aged , Racial Groups , Reference Values
20.
J Speech Lang Hear Res ; 43(1): 184-90, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10668661

ABSTRACT

The present study compares the results obtained on original and French versions of the TRQ (Tinnitus Reaction Questionnaire) initially published by Wilson, Henry, Bowen, and Haralambous (1991) in English to evaluate the psychological distress of tinnitus sufferers. Reliability and validity of the French translation were determined using data from 173 normal hearing or hearing-impaired patients with tinnitus lasting from 1 month to 41 years. They completed the translated questionnaire and a short version of the Minnesota Multiphasic Personality Inventory. The results indicated good internal consistency (Cronbach's alpha = .94), and the reliability of the French version of the TRQ was demonstrated, except for items 5 and 20. High statistically significant correlations were found between the TRQ and Depression, Psychaesthenia, and Anxiety Mini-Mult subscales. The validation demonstrates only minor effects of language. The French version of the TRQ thus is an equally valid tool as the original English version for evaluating tinnitus distress of a patient.


Subject(s)
Anxiety/etiology , Depression/etiology , Language , Stress, Psychological/etiology , Surveys and Questionnaires , Tinnitus/psychology , Adult , Anxiety/diagnosis , Depression/diagnosis , Female , Humans , Male , Middle Aged , Reproducibility of Results , Severity of Illness Index , Stress, Psychological/diagnosis , Translations
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