Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure.
(25/2410)
Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; approximately 140,000 unengaged polymerases (mainly of form IIA) were released. Only approximately 4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (approximately 110,000 molecules of form IIO) and 10,000 to 30,000 molecules of each of TFIIB, TFIIEalpha, TFIIEbeta, TFIIF-RAP74, TFIIF-RAP30, and TFIIH-MAT1 remained tightly associated with the nuclear substructure. Most proteins and run-on activity were retained when approximately 50% of the chromatin was detached with a nuclease, but approximately 45,000 molecules of bound TATA binding protein (TBP) were detached. Similar results were obtained after cross-linking living cells with formaldehyde. The results provide little support for the existence of a large pool of soluble holoenzyme; they are consistent with TBP-promoter complexes in nuclease-sensitive chromatin being assembled into preinitiation complexes attached to the underlying structure. (+info)
Evidence for tonic activation of NK-1 receptors during the second phase of the formalin test in the Rat.
(26/2410)
Behavioral, electrophysiological, and autoradiographic experiments were done to study the second nociceptive phase in the formalin test. In initial experiments, this second phase was attenuated by 1-10 mg of the NK-1 receptor antagonist CP-99,994, given subcutaneously 10, 30, or 60 min before formalin (n = 8-10) and by 20 microgram given intrathecally 20 min after formalin (n = 13); the inactive isomer CP-100,263 was ineffective. In electrophysiological experiments on single dorsal horn neurons in vivo, the excitatory responses to subcutaneous formalin injection (50 microliter, 2.5%) were attenuated by subsequent intravenously administration of the NK-1 receptor antagonist CP-96,345 (0.5 mg/kg; n = 8), given 35-40 min after formalin, but not by the inactive enantiomer CP-96,344 (0.5 mg/kg; n = 9). Finally, autoradiographic binding of exogenous [(125)I]BH-substance P in the lumbar cord was reduced at 5 and 25 min after formalin (50 microliter, 1 or 5%), with an intermediate level of reduction at 12 min. These data are interpreted as evidence that the second phase of nociceptive scores in the formalin test is attributable at least partially to tonic activation of NK-1 receptors at the spinal level, whether because of a temporally limited release of substance P, for example only during the first phase, but a slow removal or breakdown of substance P, or, more likely, because of tonic release from primary afferents throughout the second phase. Irrespective of the mechanism, it can be concluded that at least some of the persistent nociceptive effects associated with peripheral inflammation, or at least those provoked by subcutaneous injection of formalin, are mediated via continuous activation of NK-1 receptors at the level of the spinal dorsal horn; this may relate directly to mechanisms underlying prolonged nociceptive pains in humans. (+info)
Purification and properties of an esterase from the yeast Saccharomyces cerevisiae and identification of the encoding gene.
(27/2410)
We purified an intracellular esterase that can function as an S-formylglutathione hydrolase from the yeast Saccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50 degrees C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to S-formylglutathione by S. cerevisiae. (+info)
Development and testing of a microbiological assay to detect residual effects of disinfectant on hard surfaces.
(28/2410)
We describe a glucuronidase bioassay for detecting residual bactericidal activity from the use of disinfectants on hard surfaces; in this assay we used formaldehyde, ethanol, isopropanol, chlorine, and a commercial preparation containing 2-bromo-2-nitro-1, 3-propanediol. Chlorine and the commercial preparation showed bactericidal activity (53.5% and 98.2%, respectively) for a week after disinfection. (+info)
NMDA receptors mediating Fos expression in rat spinal cord induced by subcutaneous injection of formalin.
(29/2410)
AIM: To examine the effects of N-methyl-D-aspartate (NMDA) and non-NMDA receptors on noxious stimulation-induced Fos expression in the rat spinal cord. METHODS: Formalin (2%) was injected s.c. into one hindpaw of the rat. Fos expression was exhibited by immunocytochemical technique. RESULTS: Two hours after s.c. formalin, Fos-like immunoreactive (FLI) neurons were distributed mainly in medial part of the lamina I and the outer lamina II of the ipsilateral dorsal horn. dl-2-Amino-5-phosphonovalerate (APV) administered intrathecally (10 microL, 0.01, 0.1, or 1 g.L-1) before injection of formalin into a hindpaw reduced the number of FLI neurons dose-dependently in the dorsal horn (P < 0.01), while 6,7-dinitroquinoxaline-2, 3(1H,4H)-dione (DNQX) (1 g.L-1) was ineffective. CONCLUSION: NMDA receptor mediated noxious stimulation-induced Fos expression in the rat spinal cord. (+info)
Influence of refrigeration and formalin on the floatability of Giardia duodenalis cysts.
(30/2410)
Giardia duodenalis cysts obtained from fresh fecal samples, fecal samples kept under refrigeration and fecal samples treated with formalin were studied as to their floatability on sucrose solutions with the following specific gravities: 1,040 kg/m3; 1,050 kg/m3; 1, 060 kg/m3; 1,070 kg/m3; 1,080 kg/m3; 1,090 kg/m3; 1,100 kgm3; 1,150 kg/m3; 1,200 kg/m3; and 1,250 kg/m3, contained within counting-chambers 0.17 mm high. Cysts that floated on and those settled down as sediments were counted, and had their percentages estimated. Sucrose solutions of 1,200 kg/m3 specific gravity (the average specific gravity of diluting liquids employed in floatation techniques) caused to float 77.7%, 78.4% and 6.6% of the G. duodenalis cysts obtained, respectively, from fresh fecal samples, fecal samples kept under refrigeration, and fecal samples treated with formalin. Cysts obtained both from fresh fecal samples and fecal samples kept under refrigeration presented similar results concerning floatability. It was observed, however, that the treatment of feces with formalin diminished the cysts floatability under the various specific gravities studied. This results should influence, the recommendations for transport and storage of fecal samples used for parasitological coproscopy. (+info)
Infectivity of scrapie prions bound to a stainless steel surface.
(31/2410)
BACKGROUND: The transmissible agent of Creutzfeldt-Jakob disease (CJD) is not readily destroyed by conventional sterilization and transmissions by surgical instruments have been reported. Decontamination studies have been carried out thus far on solutions or suspensions of the agent and may not reflect the behavior of surface-bound infectivity. MATERIALS AND METHODS: As a model for contaminated surgical instruments, thin stainless-steel wire segments were exposed to scrapie agent, washed exhaustively with or without treatment with 10% formaldehyde, and implanted into the brains of indicator mice. Infectivity was estimated from the time elapsing to terminal disease. RESULTS: Stainless steel wire (0.15 x 5 mm) exposed to scrapie-infected mouse brain homogenate and washed extensively with PBS retained the equivalent of about 10(5) LD50 units per segment. Treatment with 10% formaldehyde for 1 hr reduced this value by only about 30-fold. CONCLUSIONS: The model system we have devised confirms the anecdotal reports that steel instruments can retain CJD infectivity even after formaldehyde treatment. It lends itself to a systematic study of the conditions required to effectively inactivate CJD, bovine spongiform encephalopathy, and scrapie agent adsorbed to stainless steel surfaces such as those of surgical instruments. (+info)
Effect of tetrahydropalmatine analogs on Fos expression induced by formalin-pain.
(32/2410)
AIM: To study the effect of tetrahydropalmatine (THP) analogs on Fos protein expression induced by formalin-pain and elucidate analgesic mechanism of THP analogs. METHODS: The pain response to Sprague Dawley rats was induced with formalin injected s.c. into the plantar surface of the right hindpaw. Fos protein expression in brain and spinal cord was investigated with immunohistochemistry. The numbers of Fos-like immunoreactive (FLI) neurons were counted with Leica Q570 image analyzer. RESULTS: In the groups of THP analogs and D2 antagonist spiperone, FLI neurons induced by intraperitoneal (i.p.) injection of THP analogs and spiperone were mainly located in the striatum and accumbens nucleus, and a few FLI neurons were also in sensorimotor cortex. In the D1 antagonist, D1 agonist, D2 agonist, saline and vehicle groups, FLI neurons were seldom seen in the striatum and accumbens nucleus. Moreover, the Fos protein expression induced by l-THP and spiperone could be prevented by the pre-treatment of the D2 agonist quinpirole but not D1 agonist SKF38393. In the formalin-pain group, FLI neurons were mainly distributed in ascending pain afferent system (APAS) and descending pain modulation system (DPMS). Following i.p. THP analogs, however, the numbers of FLI neurons induced by formalin-pain in the APAS, such as dorsal horn (mainly laminae I, II, IV-VI) were markedly decreased, while the numbers of FLI neurons in the DPMS, such as periaqueductal gray (PAG) and reticular paragigantocellular lateral nucleus (RPLN) were significantly increased. CONCLUSION: THP analogs enhanced the activity of brainstem DPMS by the blockade of D2 receptors in the striatum and accumbens nucleus, and sequentially inhibited the inputs of peripheral pain afferent message in spinal cord level. (+info)