Melatonin might be one possible medium of electroacupuncture anti-seizures.

To explore the alteration of melatonin (MT) levels in pineal, hippocampus and serum during seizure crises and electroacupuncture (EA) anti-seizures, we established a rat seizure model by microinjecting benzylpenicillin into hippocampus. EA was performed on "Fengu" (DU 16) and "Jinsuo" (DU 8) acupoints in rats. Electroencephalogram (EEG) of rats was recorded and the relative power (RP) of 1 approximately 30 HZ band of EEG was analyzed. A capillary electrophoresis-electrochemical detection method was used to determine MT contents. Our results indicated that MT level was elevated in pineal and hippocampus, and first had no change then significantly evaluated in serum during seizure crisis. The elevation of MT level was greatly potentiated with 30 min EA treatment (P<0.05). Meanwhile, the degree of seizures and the increases of EEG RP induced by seizures were significantly reduced (P<0.05). Because MT was considered as an antistressor and a natural downregulator of epileptiform activity, we postulate that the elevation in MT level during seizures may be one endogenous mechanism that counteracts convulsions and seizure-induced stress. A further elevation of MT levels with EA treatment suggests that MT might be one of the possible mediums of EA anti-seizures.

Naloxone reverses inhibitory effect of electroacupuncture on sympathetic cardiovascular reflex responses.

Acupuncture and electroacupuncture (EA) have been used in traditional Chinese medicine to treat a wide range of diseases and conditions, including angina pectoris and myocardial infarction. In a feline model of reflex-induced reversible myocardial ischemia, electrical stimulation of the median nerves to mimic EA (Neiguan acupoint) significantly improved ischemic dysfunction, secondary to an inhibitory effect of EA on reflex pressor effects evoked by bradykinin (BK). The central mechanism of EA's inhibitory effect in this model is unknown. Accordingly, in alpha-chloralose-anesthetized cats, BK (10 micrograms/ml) was applied to the gallbladder to elicit a cardiovascular reflex response that significantly (P < 0.05) increased arterial blood pressure and heart rate; normalized systolic wall thickening (%WTh) of the left ventricle, measured by ultrasonic single-crystal sonomicrometer, increased by 31 +/- 11% (P < 0.05). After ligation of a side branch of the left anterior descending coronary artery, the reflex pressor response to BK resulted in a significant decrease of %WTh (-32 +/- 6%) in the ischemic region. When bilateral EA of the Neiguan acupoints was performed, the pressor response to BK was inhibited and regional myocardial function was significantly improved (+19 +/- 20%). The inhibitory effects of EA on blood pressure and %WTh were reversed by intravenous injection of naloxone (0.4 mg/kg; n = 9) or microinjection of naloxone (10 nM in 0.1 microliter/site; n = 14) into the rostral ventrolateral medulla (rVLM). Thus %WTh with intravenous naloxone was reduced to -13 +/- 29% (P<0.05) during stimulation of the gallbladder. Our results indicate that the inhibitory effect of EA on the BK-induced pressor response and the consequent improvement of ischemic dysfunction is dependent on the activation of opioid receptors, specifically receptors located in the rVLM.

[Inhibitory effect of electroacupuncture on the cardiovascular response evoked by applying bradykinin on the gallbladder].

The effects of electroacupuncture (EA) on the pressor response and reversible myocardial dysfunction induced by application of bradykinin (BK) on the gallbladder were studied in cats anesthetized with alpha-chloralose. The cardiovascular responses evoked by application of BK included a pressor response, an increase of LVP and its dP/dtmax, tachycardia and a decrease of local wall motion of the left ventricle with a supplying branch of the left anterior descending coronary artery ligated beforehand. Following EA of bilateral Neiguan acupionts, the pressor response of BK was inhibited, while the regional left ventricle myocardial dysfunction was alleviated significantly. The effects of EA were reversed by i.v. injection of naloxone (0.4 mg/kg). Our results indicate that EA has an inhibitory effect on the BK-induced pressor and ischemic dysfunction, which may be related with endogenous opioid peptide.

A strategy for rapid, high-confidence protein identification.

A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of > 0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.

BRCA1 is a component of the RNA polymerase II holoenzyme.

The familial breast-ovarian tumor suppressor gene product BRCA1 was found to be a component of the RNA polymerase II holoenzyme by several criteria. BRCA1 was found to copurify with the holoenzyme over multiple chromatographic steps. Other tested transcription activators that could potentially contact the holoenzyme were not stably associated with the holoenzyme as determined by copurification. Antibody specific for the holoenzyme component hSRB7 specifically purifies BRCA1. Immunopurification of BRCA1 complexes also specifically purifies transcriptionally active RNA polymerase II and transcription factors TFIIF, TFIIE, and TFIIH. Moreover, a BRCA1 domain, which is deleted in about 90% of clinically relevant mutations, participates in binding to the holoenzyme complex in cells. These data are consistent with recent data identifying transcription activation domains in the BRCA1 protein and link the BRCA1 tumor suppressor protein with the transcription process as a holoenzyme-bound protein.

Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo.

Activation of eukaryotic class II gene expression involves the formation of a transcription initiation complex that includes RNA polymerase II, general transcription factors, and SRB components of the holoenzyme. Negative regulators of transcription have been described, but it is not clear whether any are general repressors of class II genes in vivo. We reasoned that defects in truly global negative regulators should compensate for deficiencies in SRB4 because SRB4 plays a positive role in holoenzyme function. Genetic experiments reveal that this is indeed the case: a defect in the yeast homologue of the human negative regulator NC2 (Dr1 x DRAP1) suppresses a mutation in SRB4. Global defects in mRNA synthesis caused by the defective yeast holoenzyme are alleviated by the NC2 suppressing mutation in vivo, indicating that yeast NC2 is a global negative regulator of class II transcription. These results imply that relief from repression at class II promoters is a general feature of gene activation in vivo.

A mammalian SRB protein associated with an RNA polymerase II holoenzyme.

A large multisubunit complex containing RNA polymerase II, general transcription factors and SRB regulatory proteins initiates transcription of class II genes in yeast cells. The SRB proteins are a hallmark of this RNA polymerase II holoenzyme as they are found only in this complex, where they contribute to the response to regulators. We have now isolated a human homologue of the yeast SRB7 gene and used antibodies against human SRB7 protein to purify and characterize a mammalian RNA polymerase II holoenzyme containing the general transcription factors TFIIE and TFIIH. This holoenzyme is more responsive to transcriptional activators than core RNA polymerase II when assayed in the presence of coactivators.

RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling.

The RNA polymerase II holoenzyme contains RNA polymerase II, a subset of general transcription factors and SRB regulatory proteins. We report here that SWI and SNF gene products, previously identified as global gene regulators whose functions include remodeling chromatin, are also integral components of the yeast RNA polymerase II holoenzyme. The SWI/SNF proteins are components of the SRB complex, also known as the mediator, which is tightly associated with the RNA polymerase II C-terminal repeat domain. The SWI/SNF components provide the holoenzyme with the capacity to disrupt nucleosomal DNA and thus facilitate stable binding of various components of the transcription initiation complex at promoters.

Association of an activator with an RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.

A kinase-cyclin pair in the RNA polymerase II holoenzyme.

The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.

A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast.

We report genetic and biochemical evidence that the RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunit complex that contains TATA-binding protein (TBP) and is an integral part of the transcription initiation complex. The isolation and characterization of extragenic suppressors of S. cerevisiae RNA polymerase II CTD truncation mutations led us to identify SRB2, SRB4, SRB5, and SRB6 as genes involved in CTD function in vivo. SRB2 was previously isolated and shown to encode a 23 kd TBP-binding protein. The four SRB proteins and a portion of cellular TBP are components of a high molecular weight multisubunit complex that is tightly bound to RNA polymerase II. This SRB-TBP complex binds specifically to recombinant CTD protein. In vitro transcription and template commitment assays confirm that SRB2 and SRB5 are components of a functional preinitiation complex and are required for efficient transcription initiation.