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Query: UNIPROT:P06889 (Mol)
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Duchenne and mdx muscle tissues lack dystrophin where it normally interacts with glycoproteins in the sarcolemma. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myotubes and is correlated with abnormally active calcium-specific leak channels in dystrophic myotubes. We fused Duchenne human and normal mouse myoblasts and identified heterokaryon myotubes by Hoechst 33342 staining to measure the degree to which dystrophin introduced by normal nuclei could incorporate throughout the myotube at the sarcolemma and restore normal calcium homeostasis. Dystrophin expression in myotubes was determined by immunofluorescence and confocal laser scanning microscopy. Dystrophin was expressed at the sarcolemma in normal mouse and heterokaryon myotubes, but not in Duchenne myotubes. In heterokaryons, extensive dystrophin localization occurred at the sarcolemma even where only Duchenne nuclei were present, indicating that dystrophin does not exhibit nuclear domains. Heterokaryon, normal mouse and Duchenne myotube [Ca2+]i was measured using fura-2 and fluorescence ratio imaging. Heterokaryon and normal mouse myotubes were found to maintain similar levels of [Ca2+]i. In contrast, Duchenne myotubes had significantly higher [Ca2+]i (p < 0.001). Furthermore, the ability of heterokaryons to maintain normal [Ca2+]i did not depend on greater numbers of normal nuclei than Duchenne being present in the myotube. These results support the view that dystrophin expression in heterokaryons allows for efficient control of [Ca2+]i.
Mol Biol Cell 1993 Sep
PMID:Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control. 825 98

After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN-type and compact in SN-type oocytes. Using video-enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics.
Mol Reprod Dev 1993 Sep
PMID:Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics. 839 31

This study examined the competence of oocytes from the tammar wallaby, Macropus eugenii to mature in vitro. Oocytes were collected from follicles > 1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35 degrees C in 5% CO2 in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml-1 PMSG or 10 micrograms ml-1 porcine luteinizing hormone (LH) + 10 micrograms ml-1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (< 1.5-mm-diameter) and large (> or = 1.5 mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1993 Mar
PMID:In vitro maturation of oocytes from a marsupial, the tammar wallaby (Macropus eugenii). 847 Dec 56

The inoculation of pea endocarp tissue with the bean pathogen Fusarium solani f. sp. phaseoli results in a non-host resistance response causing a complete cessation of fungal growth within 6 to 8 h. In addition to previously reported elicitation by chitosan, we now report that components of this response are also induced by a DNase released from this fungus. A single band of protein corresponding with DNase activity elicits phytoalexin production and the accumulation of RnA homologous with the pathogenesis-related (PR) genes DRR49, DRR206, and DRR230. Both the enzyme activity and the eliciting potential of the Fusarium DNase (Fsp DNase) are heat stable but susceptible to digestion by proteinase K. Fsp DNase mimics the intact fungus in inducing resistance against F. solani f. sp. pisi. Also, Fsp DNase causes similar cytologically detectable changes in pea tissue, such as increasing hypersensitive discoloration and diminishing fluorescence of Hoechst 33342-stained nuclei and fluorescein diacetate stained cells.
Mol Plant Microbe Interact
PMID:Fusarium solani DNase is a signal for increasing expression of nonhost disease resistance response genes, hypersensitivity, and pisatin production. 866 96

To facilitate the study of interactions between equine spermatozoa and homologous oviduct epithelial cells, we developed an assay to count labelled spermatozoa bound to oviduct epithelial cell (OEC) monolayers and used the assay to compare the binding ability of spermatozoa from different stallions. Washed spermatozoa from three stallions were incubated with the fluorochrome Hoechst 33342 (5 micrograms/ml) for 1 min. Spermatozoa were then layered over confluent monolayers of oviduct epithelial cells in 2 cm2 culture wells. Coculture treatments comprised five concentrations of spermatozoa (10(5), 5 x 10(5), 10(6), 2.5 x 10(6), and 5 x 10(6) per well). Cocultures were incubated for 30 min before unattached spermatozoa were aspirated in coculture supernatant. Fluorescent videoimages of attached spermatozoa were digitized, and attached spermatozoa were counted by image processing and analysis. Four wells (replicates) of each concentration were allocated within each ejaculate, and ejaculates were blocked by stallion for ANOVA. The total number of spermatozoa bound was not different between replicate wells (P > 0.1). Stallion, ejaculate, concentration, and all higher level interactions influenced total spermatozoa bound (P < 0.00001). Coefficients of variation between replicates were lowest for inseminate concentrations between 10(6) and 5 x 10(6) spermatozoa per well. Within the ejaculate, a log linear relationship exists between the number of bound spermatozoa and a spermatozoal concentration of the inseminate between 5 x 10(5) and 5 x 10(6) spermatozoa per well. This assay provides a reliable method of determining numbers of spermatozoa bound to somatic cells in vitro. Furthermore, differences exist in the ability of spermatozoa from different stallions to bind OEC monolayers.
Mol Reprod Dev 1996 Jan
PMID:Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro. 872 Jan 13

Two cell types, HL60 human promyelocytic leukemia cells and CD34+ human bone marrow progenitor cells, were used as model systems to explore a possible role for apoptosis in the myelotoxicity of the phenolic metabolites of benzene. HL60 cells were treated with either phenol, catechol, hydroquinone, or 1,2,4-benzenetriol and then stained with Hoechst 33342 and propidium iodide and subjected to fluorescent microscopy. Cells with nuclear condensation and fragmentation were scored as apoptotic, and etoposide (40 microM) was used as a positive control. Catechol, 1,2,4-benzenetriol, and hydroquinone induced marked time- (0-24 hr) and concentration- (25-100 microM) dependent apoptosis, whereas phenol (750 microM) did not. Under these conditions, no significant necrosis was observed. The induction of apoptosis was confirmed by internucleosomal cleavage of DNA, assessed by agarose gel electrophoresis. CD34+ cells treated with etoposide (40 microM) or hydroquinone (50 microM) for 18 hr were stained and subjected to fluorescent microscopy as above. The percentage of cells exhibiting nuclear condensation and/or fragmentation as well as high intensity staining significantly increased in both cases. The induction of apoptosis was confirmed using a terminal deoxynucleotidyl transferase assay. These data show that apoptosis can be induced in both HL60 and CD34+ human bone marrow progenitor cells by benzene metabolites. The ability of phenolic metabolites of benzene to induce apoptosis in human bone marrow progenitor cells may contribute to benzene myelotoxicity.
Mol Pharmacol 1996 Sep
PMID:Induction of apoptosis by benzene metabolites in HL60 and CD34+ human bone marrow progenitor cells. 879 1

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0-100 microg/ml) and apigenin (250 microM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10-400 microg/ml). Sperm motility following exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovulated hamster OCCs for 30 min at 37 degrees C. The addition of heparin, apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity (P < .01). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1-100 microg/ml), apigenin (250 microM), or R10 antibody (Ig fraction, 10-400 microg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P < .01). These results suggest that macaque sperm PH-20-derived hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction.
Mol Reprod Dev 1997 Mar
PMID:Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay. 904 Nov 43

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 microg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38 degrees C, 4 hr), flow sorting, cooling (to 15 or 5 degrees C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C (30.4, 48.5, 21.1%) than in those cooled to 15 degrees C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively).
Mol Reprod Dev 1997 Mar
PMID:Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation. 904 Nov 45

Repopulation by donor cells of a bone marrow ablated by irradiation is now recognized to proceed in two phases: initial repopulation that may be temporary followed by permanent engraftment of longterm repopulating cells (LTRC). While a single LTRC has been shown to be capable of restoring the entire lymph-hemopoietic system of an irradiated animal, the identity of the temporary repopulating cells has not been established unequivocally. We used the results of transplantation of subpopulations successively enriched for LTRC and containing varying numbers of CFU-S-12 (colony-forming units in the spleen at day 12 post transplantation) and progenitors to determine the likely cell type and number of cells needed for initial survival after radiation. Subpopulations from untreated and 5-fluorouracil-treated mice were discriminated on the basis of antibody reactivity, Hoechst 33342 and rhodamine 123 fluorescence intensity and light scattering properties. The minimum rescue inocula varied greatly in CFU-GEMM, BFU-E and CFU-GM content. One to two CFU-S-12 were uniformly present in all isolated suspensions that rescued 50% of lethally irradiated animals. In view of the known average seeding efficiency of CFU-S, our studies suggest that transfusion of 10-20 CFU-S day 12/13 is responsible for radioprotection. Evidence that multiple CFU-S day 12/13 are needed for initial repopulation is also supported by quantitative estimates of the number of mature cells that can be produced by CFU-S. Transfusion of a single CFU-S day 12/13 can be shown to be grossly inadequate to provide the number of peripheral blood cells needed to ameliorate the severe pancytopenia following lethal irradiation by day 12-14. Our data also indicate that 5-fluorouracil-treated marrow subpopulations appear inferior to untreated subpopulations in their ability to contribute to initial repopulation when transfused at low cell doses into lethally irradiated recipients.
Blood Cells Mol Dis 1997 Aug
PMID:Rescue from lethal irradiation correlates with transplantation of 10-20 CFU-S-day 12. 923 54

Several procedures exist for processing sperm cells for sex preselection. Flow cytometric separation using the fluorochrome stain Hoechst 33342, chemically known as bisbenzimide, is the most promising. The objective of this study was to determine the effect of bisbenzimide on spermatozoa assessed by means of the sperm survival test and to analyse the beta-globin gene in sperm DNA after exposure to increasing concentrations of bisbenzimide. Donor (n = 16) sperm specimens were pooled and washed in a discontinuous Percoll gradient 95:47%, divided and incubated in tubes containing bisbenzimide at concentrations 0 (control), 0.9, 9, 90, 900 and 9000 microM at 25 degrees C and scanned on a computer-aided sperm motility analyser at 0, 1, 4 and 24 h. Spermatozoa were also incubated in a known mutagen, ethidium bromide, as positive control. After 24 h of incubation, the treated sperm cells were processed through DNA extraction and polymerase chain reaction (PCR) performed with primers targeting the beta-globin gene. The amplified DNA products were analysed for evidence of mutation in 5% polyacrylamide gel electrophoresis and 20:80 denaturing gradient gel electrophoresis (DGGE) and further confirmed in 30:40 DGGE. The results showed complete cessation of motility in sperm incubated in the presence of 900 microM or higher concentrations of bisbenzimide. The beat cross frequency sperm parameter was significantly different at the 90 microM or higher concentration of bisbenzimide compared with the control. At concentrations < 900 microM bisbenzimide, there were no differences in the remaining sperm kinematic parameters (percentage rapid progressive, percentage total progressive, sperm velocities, linearity, straightness, amplitude of lateral head displacement and percentage hyperactive motility). PCR and DGGE analyses of spermatozoa treated with bisbenzimide showed no evidence of mutation in the representative region of the beta-globin gene at concentrations < 900 microM. The data suggest an inhibitory effect of bisbenzimide on human sperm motility at 900 microM or higher concentrations of bisbenzimide. The decrease in sperm motility and rapid progression were not due to changes in pH. Point mutation in the representative region of the beta-globin gene in human spermatozoa was detected only at high concentrations (> or = 900 microM) of bisbenzimide. The data suggest that incubating sperm in low concentrations of bisbenzimide (< 90 microM) for up to 24 h does not significantly affect all the sperm kinematic parameters including the beat cross frequency parameter when compared with the control.
Mol Hum Reprod 1996 Sep
PMID:Analysis of the flow cytometer stain Hoechst 33342 on human spermatozoa. 923 86


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