Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously we demonstrated that lines of transgenic mice carrying the herpes simplex type 1 virus thymidine kinase (HSV1-tk) reporter gene are male-sterile. Ectopic transcription of the HSV1-tk reporter in the testis was initiated downstream of the normal translation initiation codon and truncated proteins consistent with translational initiation at the second and third ATG codons were synthesized. Here we describe the effects on fertility 1) of converting the second and third ATG codons of the HSV1-tk reporter to CTG codons and 2) of utilizing the HSV type 2 thymidine kinase (HSV2-tk) reporter gene, in which the second ATG codon is located downstream of the ATP-binding pocket of the enzyme. Both reporters were coupled to the bovine thyroglobulin promoter (bTG-tk1 alpha and bTG-tk2 transgenes). The level of ectopic expression of these transgenes in the testis, relative to expression in the thyroid, was one to two orders of magnitude less than that of bTG-tk1. Sixty percent of male founders carrying the bTG-tk1 alpha and bTG-tk2 transgenes were fertile but did not transmit the transgene. In contrast, most males from subsequent generations were fertile and transmitted the transgenes at the expected frequency. This difference between founder males and male descendants is also observed with certain constructs in which the HSV1-tk reporter is coupled to other promoters. We attribute the effect to mosaicism among male founders, leading to competition between transgenic and nontransgenic spermatozoa and/or spermatogenic precursor cells and resulting in a lack of fertilization by transgenic sperm that would successfully fertilize eggs in the absence of competition.
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PMID:Different transmission rates of herpesvirus thymidine kinase reporter transgenes from founder male parents and male parents of subsequent generations. 757 10

The herpes simplex type 1 virus thymidine kinase (HSV1-TK) reporter gene was coupled to a bovine thyroglobulin promoter (TG-tk construct). Within the thyroid glands of transgenic mice expression was confined to thyroid follicle cells. Infusion of Ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) to 8 to 12 week transgenic females led to the complete loss of thyroid HSV1-TK activity (at 3 to 4 days) and thyroid follicles (between 7 and 14 days). During the first 5 days of treatment a single reciprocal oscillation in circulating thyroxine (T4) and TSH levels occurred. By 14 days the circulating tri-iodothyronine (T3) and T4 levels of all treated animals were below the detection limits of the assays, while TSH levels were elevated ten-fold and continued to increase thereafter. During 14 days of treatment the thyroids regressed, protein content fell by 80-90% and the C cells, normally dispersed within the central region of each gland, came together in aggregates. Pituitary GH levels in females rose and fell back to normal within 14 days and between 14 and 28 days fell to a level comparable with that of GH-deficient lit/lit mice. The levels of hepatic GH receptor mRNA and the predominant 6.6 kb T3 receptor mRNA were unaffected by thyrocyte ablation. Thyrocyte ablation had no effect on the level of prolactin (Prl) receptor mRNA in females, but increased Prl receptor mRNA levels in males and eliminated group 1 major urinary protein (MUP).mRNA in females. T4 replacement reversed the effects of thyrocyte ablation on MUP mRNA in females and on Prl receptor mRNA in males.2+ Despite the many physiological changes induced by thyrocyte ablation, ablated mice have been maintained for up to 1 year without thyroid hormone supplementation. T4-deficient females were normally fertile and carried pups to term. Although transgenic males expressed HSV1-TK ectopically in spermatids and spermatozoa at levels similar to thyrocyte levels, a rate of Ganciclovir infusion which successfully ablated the thyrocytes did not affect the testis. As an alternative to infusion by minipump, thyrocyte ablation could be achieved by 6 twice-daily injections of Ganciclovir, at a level of 112 micrograms Ganciclovir/g body weight per day, and fetuses in utero could be thyrocyte ablated by administering 50 or 15 micrograms/g body weight per day to pregnant females between days 14 and 18 of gestation. These data demonstrate the potential value of transgenic thyrocyte ablation in the study of the effects of thyroid hormone deprivation.
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PMID:Consequences of thyroid hormone deficiency induced by the specific ablation of thyroid follicle cells in adult transgenic mice. 796 9

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at -20 degrees C until use. When the sperm heads were aspirated into injection pipettes 7-10 microm in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2-4 microm in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague-Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.
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PMID:Offspring derived from intracytoplasmic injection of transgenic rat sperm. 1205 55

Octoxynols are ethoxylated alkylphenols in which the size of the molecule is related to the number of moles of ethylene oxide used in synthesis. Reactions are performed at elevated temperature, under pressure, and in the presence of NaOH. It is possible that the synthesis may leave trace amounts of ethylene oxide, 1,4-dioxane, and unreacted C9 phenols. Octoxynols of various chain lengths as well as octoxynol salts and organic acids function in cosmetics either as surfactants--emulsifying agents, surfactants--cleansing agents, surfactant--solubilizing agents, or surfactants--hydrotropes in a wide variety of cosmetic products at concentrations ranging from 0.0008% to 25%, with most less than 5.0%. The octoxynols are chemically similar to nonoxynols, the safety of which were previously considered. Long-chain nonoxynols (9 and above) were considered safe as used, whereas short-chain nonoxynols (8 and below) were considered safe as used in rinse-off products and safe at concentrations less than 5% in leave-on formulations. Acute exposure of hamsters to Octoxynol-9 by bronchopulmonary lavage produced pneumonia, pulmonary edema, and intra-alveolar hemorrhage. Octoxynol-9 at doses over 1 g/kg was toxic in rats and in mice in acute oral toxicity studies. No significant effects were noted in short-term oral studies of Octoxynol-9 in rats, in subchronic oral studies of Octoxynol-40 in rats and dogs, or in chronic oral studies of Octoxynol-40 in rats. The intraperitoneal LD50 of Octoxynol-9 in rats and mice was around 100 mg/kg. In skin irritation studies, octoxynols ranged from nonirritating to moderately irritating. Octoxynols were not ocular irritants in one rabbit study, but in others there was ocular irritation. No immune system toxicity in CF-1 female mice was noted following the intraperitoneal injection of Octoxynol-9 followed by subcutaneous immunization with sheep red blood cells (SRBCs). Octoxynol-9 produced no humoral and cell-mediated immune responses, or autoimmune response in mice. In the Ames test, Octoxynol-1 was not mutagenic with and without metabolic activation nor was Octoxynol-9 clastogenic. Results for Octoxynol-9 were negative in the following assays: unscheduled DNA synthesis, hypoxanthine guanine phosphoribosyl transferase mutation assay, malignant transformation assay, DNA alkaline unwinding test, and mouse lymphoma thymidine kinase locus forward mutation assay. Ethoxylated alkylphenols are generally considered to be estrogenic in that they mimic the effects of estradiol. Dermal exposure at three dose levels of rats to Octoxynol-9 failed to induce any malformations by category (external, visceral, or skeletal) or by individual anatomical location that were different from controls at statistically significant level. An increased incidence of a vestigial thoracic rib was observed in all dose groups. Octoxynol-9 also did not induce developmental toxicity (number of viable litters, liveborn per litter, percentage survival, birth weight per pup, and weight gain per pup) in female specific pathogen-free CD-1 mice dosed daily by gavage on gestation days 6 through 13. No reproductive toxicity was seen in male albino rats which received 5% Octoxynol-40 in the diet daily for 3 months; however, in an in vitro test, Octoxynol-9 (0.24 mg/ml) totally immobilized all human spermatozoa within 20 s. Women who used Nonoxynol-9 or Octoxynol-9 as spermicides, but who did become pregnant, did not have an increase in the overall risk of fetal malformations. In a human skin irritation study, formulations containing 2.0% Octoxynol-9 were classified as moderately irritating and minimally irritating, respectively, in a 24-h single-insult, occlusive patch test. Octoxynol-9 (1.0%) was classified as a nonirritant in a clinical study of nine subjects patch tested for 4 consecutive days. The skin sensitization potential of Octoxynols-1, -3, -5, -9, and -13 was evaluated using 50 subjects. Octoxynol-1 induced sensitization in two subjects; all other results were negative. No sensitization was observed in the following studies: 8.0% Octoxynol-9 in 103 subjects, 0.5% Octoxynol-9 in 102 subjects, and 0.1% Octoxynol-9 in 206 subjects. Concerns about even trace levels of 1,4-dioxane, ethylene oxide, or unreacted C9 led to the recommendation that levels be limited. Concerns about the ocular irritancy of short-chain octoxynols led to a recommendation that they should not be used in products that will be used in the area surrounding the eyes. A limitation on the use concentration for short-chain octoxynols (8 and below) arose from consideration of the skin sensitization potential of octoxynols and the recognition that the short-chain octoxynols could be absorbed into the skin more than the long-chain octoxynols. Overall, based on the available data, it was concluded that long-chain octoxynols (9 and above) are safe as used, whereas short-chain octoxynols (8 and below) are safe as used in rinse-off products and safe at concentrations less than 5% in leave-on formulations.
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PMID:Final report on the safety assessment of octoxynol-1, octoxynol-3, octoxynol-5, octoxynol-6, octoxynol-7, octoxynol-8, octoxynol-9, octoxynol-10, octoxynol-11, octoxynol-12, octoxynol-13, octoxynol-16, octoxynol-20, octoxynol-25, octoxynol-30, octoxynol-33, octoxynol-40, octoxynol-70, octoxynol-9 carboxylic acid, octoxynol-20 carboxylic acid, potassium octoxynol-12 phosphate, sodium octoxynol-2 ethane sulfonate, sodium octoxynol-2 sulfate, sodium octoxynol-6 sulfate, and sodium octoxynol-9 sulfate. 1516 38

A transgenic rat was established using the construct of porcine FSHbeta subunit promoter, the -852/+10 bp region, fused to a Herpes simplex virus thymidine kinase (HSV-TK) gene. Integration of the transgene was confirmed by PCR of tail DNA. RT-PCR of total RNAs of the pituitary, gonad, cerebellum, liver, kidney, adrenal gland, prostate, and uterus revealed that FSHbeta was only expressed in the pituitary. Analysis of the expression of reporter gene, HSV-TK, using two specific primer sets revealed that different transcripts were present in the pituitary and testis. The transcript initiated at the transcription initiation site of the porcine FSHbeta gene was detected in the pituitary, and another within the TK gene was found in the testis, indicating ectopic testis-specific expression. Immunohistochemistry of the pituitary glands of the transgenic rats for FSH and HSV-TK demonstrated that the FSH-producing cells also produced HSV-TK. The results indicated that the -852/+10 bp region of the FSHbeta promoter contains an element(s) that determines the tissue-specific expression. We succeeded in producing FSHbeta promoter-driven HSV-TK transgenic rats and were the first time to do so using an animal other than the mouse. The transgenic rats show male infertility that involves abnormal spermatogenesis. We also observed a decrease in the weight of the testis and epididymis, and both motile and living spermatozoa were absent in the epididymis. Consequently, the FSHbeta-HSV-TK transgenic rat will provide a useful model for studies on FSH function and male infertility.
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PMID:Expression of porcine FSHbeta subunit promoter-driven herpes simplex virus thymidine kinase gene in transgenic rats. 1713 9

Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322-base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 +/- 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non-cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.
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PMID:Assessment of viral presence in semen and reproductive function of frozen-thawed spermatozoa from Pallas' cats (Otocolobus manul) infected with feline herpesvirus. 1731 33

Transgenic rats show spermatid-specific ectopic expression of the reporter gene, herpes simplex virus type1 thymidine kinase (HSV1-TK), in the testes and have demonstrated male infertility. However, the disruption of spermatogenesis and the underlying molecular mechanisms in these transgenic animals have not been well clarified. In this study, light and electron microscopic observations were performed to characterize the morphological changes in the testes. To explore the molecular mechanisms of male infertility in the HSV1-TK transgenic rat, cDNA microarray and quantitative real-time PCR analyses were performed. The seminiferous tubules of 3-month-old transgenic rats showed morphological alterations including seminiferous epithelial sloughing, vacuolization, and degeneration of spermatogenic cells, suggesting a failure of Sertoli-germ cell interaction. Components of the epididymal lumen from transgenic rats included abnormal spermatozoa, degenerating round spermatids and abnormal elongated spermatids indicating an appearance of direct impairment of spermiogenesis. cDNA microarray and real-time PCRanalyses revealed significant changes (P<0.05) in the gene expression level in six genes, testin, versican, mamdc1, fgf7, ostf1 and cnot7. Among them, testin drew most of our attention, since the testin gene is a sensitive marker for disruption of Sertoli-germ cell adhesion. Thus, our results suggest that the accumulation of HSV1-TK in the spermatids not only directly interferes with spermiogenesis but also disrupts spermatogenesis through a disruption of Sertoli-germ cell adhesions. It is important to explore the testicular actions of the HSV1-TK protein in transgenic experimental models and thereby gain clues to find an appropriate treatment for HSV-infected patients exhibiting human male infertility, as has been recently observed.
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PMID:Accumulated HSV1-TK proteins interfere with spermatogenesis through a disruption of the integrity of Sertoli-germ cell junctions. 2278 19

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