TAFII55 binding to TAFII250 inhibits its acetyltransferase activity.

The general transcription factor, TFIID, consists of the TATA-binding protein (TBP) associated with a series of TBP-associated factors (TAFs) that together participate in the assembly of the transcription preinitiation complex. One of the TAFs, TAF(II)250, has acetyltransferase (AT) activity that is necessary for transcription of MHC class I genes: inhibition of the AT activity represses transcription. To identify potential cellular factors that might regulate the AT activity of TAF(II)250, a yeast two-hybrid library was screened with a TAF(II)250 segment (amino acids 848-1279) that spanned part of its AT domain and it's the domain that binds to the protein, RAP74. The TFIID component, TAF(II)55, was isolated and found to interact predominantly with the RAP74-binding domain. TAF(II)55 binding to TAF(II)250 inhibits its AT activity. Importantly, the addition of recombinant TAF(II)55 to in vitro transcription assays inhibits TAF(II)250-dependent MHC class I transcription. Thus, TAF(II)55 is capable of regulating TAF(II)250 function by modulating its AT activity.

Extensive interactions between HIV TAT and TAF(II)250.

The HIV transactivator, Tat, has been shown to be capable of potent repression of transcription initiation. Repression is mediated by the C-terminal segment of Tat, which binds the TFIID component, TAF(II)250, although the site(s) of interaction were not defined previously. We now report that the interaction between Tat and TAF(II)250 is extensive and involves multiple contacts between the Tat protein and TAF(II)250. The C-terminal domain of Tat, which is necessary for repression of transcription initiation, binds to a segment of TAF(II)250 that encompasses its acetyl transferase (AT) domain (885-1034 amino acids (aa)). Surprisingly, the N-terminal segment of Tat, which contains its activation domains, also binds to TAF(II)250 and interacts with two discontinuous segments of TAF(II)250 located between 885 and 984 aa and 1120 and 1279 aa. Binding of Tat to the 885-984 aa segment of TAF(II)250 requires the cysteine-rich domain of Tat, but not the acidic or glutamine-rich domains. Binding by the N-terminal domain of Tat to the 1120-1279 aa TAF(II)250 segment does not involve the acidic, cysteine- or glutamine-rich domains. Repression of transcription initiation by Tat requires functional TAF(II)250. We now demonstrate that transcription of the HIV LTR does not depend on TAF(II)250 which may account for its resistance to Tat mediated repression.

Transcriptional coactivator, CIITA, is an acetyltransferase that bypasses a promoter requirement for TAF(II)250.

The CIITA coactivator is essential for transcriptional activation of MHC class II genes and mediates enhanced MHC class I transcription. We now report that CIITA contains an intrinsic acetyltransferase (AT) activity that maps to a region within the N-terminal segment of CIITA, between amino acids 94 and 132. The AT activity is regulated by the C-terminal GTP-binding domain and is stimulated by GTP. CIITA-mediated transactivation depends on the AT activity. Further, we report that, although constitutive MHC class I transcription depends on TAF(II)250, CIITA activates the promoter in the absence of functional TAF(II)250.

TAF(II)250-independent transcription can be conferred on a TAF(II)250-dependent basal promoter by upstream activators.

TAF(II)250, a component of the general transcription factor, TFIID, is required for the transcription of a subset of genes, including those involved in regulating cell cycle progression. The tsBN462 cell line, with a temperature-sensitive mutation of TAF(II)250, grows normally at 32 degrees C, but when grown at 39.5 degrees C, it differentially arrests transcription of many, but not all, genes. The present studies examine the basis for the requirement for TAF(II)250. We show that the basal promoter of a major histocompatibility complex class I gene requires TAF(II)250. This dependence can be overcome by select upstream regulatory elements but not by basal promoter elements. Thus, the coactivator CIITA rescues the basal promoter from the requirement for TAF(II)250, whereas introduction of a canonical TATAA box does not. Similarly, the SV40 basal promoter is shown to require TAF(II)250, and the presence of the 72-base pair enhancer overcomes this requirement. Furthermore, the SV40 72-base pair enhancer when placed upstream of the basal class I promoter renders it independent of TAF(II)250. These data suggest that the assembly of transcription initiation complexes is dynamic and can be modulated by specific transcription factors.

Localization and characterization of speech arrest during transcranial magnetic stimulation.

OBJECTIVE: To determine the anatomic and physiologic localization of speech arrest induced by repetitive transcranial magnetic stimulation (rTMS), and to examine the relationship of speech arrest to language function. METHODS: Ten normal, right-handed volunteers were tested in a battery of language tasks during rTMS. Four underwent mapping of speech arrest on a 1 cm grid over the left frontal region. Compound motor action potentials from the right face and hand were mapped onto the same grid. Mean positions for speech arrest and muscle activation were identified in two subjects on 3-dimensional MRI. RESULTS: All subjects had lateralized arrest of spontaneous speech and reading aloud during rTMS over the left posterior-inferior frontal region. Writing, comprehension, repetition, naming, oral praxis, and singing were relatively spared (P < .05). Stimulation on the right during singing abolished melody in two subjects, but minimally affected speech production. The area of speech arrest overlay the caudal portion of the left precentral gyrus, congruous with the region where stimulation produced movement of the right face. CONCLUSIONS: The site of magnetic speech arrest appears to be the facial motor cortex. Its characteristics differ from those of classic aphasias, and include a prominent dissociation among different types of speech output.

Upstream stimulatory factor regulates major histocompatibility complex class I gene expression: the U2DeltaE4 splice variant abrogates E-box activity.

The tissue-specific expression of major histocompatibility complex class I genes is determined by a series of upstream regulatory elements, many of which remain ill defined. We now report that a distal E-box element, located between bp -309 and -314 upstream of transcription initiation, acts as a cell type-specific enhancer of class I promoter activity. The class I E box is very active in a neuroblastoma cell line, CHP-126, but is relatively inactive in the HeLa epithelial cell line. The basic helix-loop-helix leucine zipper proteins upstream stimulatory factor 1 (USF1) and USF2 were shown to specifically recognize the class I E box, resulting in the activation of the downstream promoter. Fine mapping of USF1 and USF2 amino-terminal functional domains revealed differences in their abilities to activate the class I E box. Whereas USF1 contained only an extended activation domain, USF2 contained both an activation domain and a negative regulatory region. Surprisingly, the naturally occurring splice variant of USF2 lacking the exon 4 domain, U2DeltaE4, acted as a dominant-negative regulator of USF-mediated activation of the class I promoter. This latter activity is in sharp contrast to the known ability of U2DeltaE4 to activate the adenovirus major late promoter. Class I E-box function is correlated with the relative amount of U2DeltaE4 in a cell, leading to the proposal that U2DeltaE4 modulates class I E-box activity and may represent one mechanism to fine-tune class I expression in various tissues.

HIV-1 tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes.

HIV Tat, a transactivator of viral transcription, represses transcription of major histocompatibility (MHC) class I genes. Repression depends exclusively on the C-terminal domain of Tat, although the mechanism of this repression has not been known. We now show that repression results from the interaction of Tat with the TAFII250 component of the general transcription factor, TFIID. The C-terminal domain of Tat binds to a site on TAFII250 that overlaps the histone acetyl transferase domain, inhibiting TAFII250 histone acetyl transferase activity. Furthermore, promoters repressed by Tat, including the MHC class I promoter, are dependent on TAFII250 whereas those that are not repressed by Tat, such as SV40 and MuLV promoters, are independent of functional TAFII250. Thus, Tat repression of MHC class I transcription would be one mechanism by which HIV avoids immune surveillance.

Presentation of narcolepsy after 40.

To advance understanding of the clinical spectra of narcolepsy, we retrospectively reviewed the histories and clinical and polysomnographic features of 41 consecutive patients in whom this diagnosis was established in our center over 3 years. A total of 51% presented after the age of 40 years. Among the older patients, three subpopulations were noted: 1) narcolepsy/cataplexy with presentation delayed because of mild disease severity or misdiagnosis; 2) narcolepsy/cataplexy with diagnosis delayed until late-life expression of cataplexy; and 3) narcolepsy lacking cataplexy with later-life onset of excessive daytime sleepiness. Clinical, polysomnographic, and multiple sleep latency test assessments of rapid eye movement sleep dyscontrol and sleepiness were unrelated to age. This analysis identified older patients lacking cataplexy as the least severely affected narcoleptic subgroup. Narcolepsy, a continuum of phenotypes and severities that masks its recognition, should be considered in the differential diagnosis of sleepiness or transient loss of muscle tone in older patients.

Optimum stimulus parameters for lateralized suppression of speech with magnetic brain stimulation.

Rapid-rate transcranial magnetic brain stimulation produces lateralized suppression of speech output over the frontal lobe, consistent with cerebral dominance for language. But the sensitivity of magnetic speech localization has been limited, and reports are imprecise concerning the amount of discomfort involved. Using a focal magnetic coil, we evaluated the effectiveness and pain of stimulation at different intensities, orientations, and repetition rates (2 to 32 Hz) in six normal volunteers. We obtained complete and clearly lateralized speech arrest in all subjects. The best ratio of efficacy to pain occurred using slower repetition rates of 4 to 8 Hz with a horizontal alignment of the induced electric field. Lower stimulation frequency also allowed clearer distinction between speech arrest and dysarthria from tonic contraction of cranial muscles. The relative comfort and safety of stimulation at 4 Hz should allow more widespread use of magnetic speech localization in clinical and research applications.

Active repression of major histocompatibility complex class I genes in a human neuroblastoma cell line.

Human neuronal cells express neither major histocompatibility complex (MHC) class I RNA nor cell surface molecules but can be induced to do so by various cytokines. In the present studies, we report that expression of MHC class I in a neuroblastoma cell line, CHP-126, is actively repressed. This repression is mediated by the combined effects of a series of upstream silencer elements. Removal of the silencers reveals not only an active promoter element but also the presence of an active enhancer. Four silencers have been identified and shown to have distinct sequences, binding factors, and patterns of function. One element is located between -724 and -697 base pairs (bp) and corresponds to a silencer involved in tissue-specific regulation of class I gene expression. Three additional elements occur between -503 and -402 bp. One of these corresponds to a c-jun responsive element. Neither of the remaining elements corresponds to DNA sequences known to regulate expression of other genes. These data demonstrate that MHC class I expression normally is actively repressed in neuronal cells and suggest a model of rapid and specific triggering of class I in neuronal cells in response to infection.

Evaluation of 1H magnetic resonance spectroscopic imaging as a diagnostic tool for the lateralization of epileptogenic seizure foci.

The purpose of this study was to assess whether a visual examination of 1H spectroscopic images could correctly lateralize patients with intractable temporal lobe epilepsy. 20 patients with intractable temporal lobe epilepsy and 10 volunteers were included in this study. Spectroscopic images were analysed using a protocol based on visual inspection. Images of the metabolites N-acetyl aspartate (NAA), choline (Cho), creatine (Cr) and lactate were obtained from a transverse plane oriented along the sylvian fissure. Images from each individual were evaluated independently by six reviewers. Results of the lateralization procedure obtained from the visual examinations were compared with those obtained from quantitative analysis of the spectra and with those obtained by magnetic resonance imaging (MRI), positron emission tomography (PET), neuropsychological examinations, and electroencephalographic (EEG) recordings. NAA images were found to be the most effective, amongst metabolite images, in lateralizing the epileptogenic lobe. Using the site selected for resection as the definition of the correct lateralization, 70% of the patients who underwent temporal lobectomy were correctly lateralized by the majority of the examiners using the visual inspection protocol. Based on the results of this study it is concluded that visual examination of 1H spectroscopic images is potentially valid in lateralizing patients with intractable temporal lobe seizures. Confidence in the visual interpretation increased as the difference in NAA signal intensity between the temporal lobes increased. The threshold above which the majority of the examiners correctly lateralized the patients was approximately 15% in NAA signal loss in the ipsilateral lobe.

Repression of the nonclassical MHC class I gene H2-M1 by cis-acting silencer DNA elements.

H2-M1 is a non-classical major histocompatibility complex (MHC) class I gene that is highly divergent from classical class I genes; M1 was the first gene in the recently classified M region of the mouse MHC to be cloned. Although the M1 DNA sequence contains normal splice sites, open reading frames within its exons, and a recognizable promoter, no M1 transcripts were detected in various healthy mouse tissues. However, M1 transcripts were detected in transfected L cells and in vivo in brains of M1 transgenic mice, albeit at very low levels, and the level of expression is correlated with transgene copy number. Analysis of the M1 promoter region identified a competent promoter capable of directing transcription, but whose expression is repressed by two strong upstream silencer elements, one mapping between -184 base pairs (bp) and -266 bp and the other between -1149 bp and -1702 bp. These studies suggest that M1 expression is highly regulated and restricted either temporally or to a very limited number of cell types.

Unique expression of major histocompatibility complex class I proteins in the absence of glucose trimming and calnexin association.

Recent evidence indicates that efficient expression of major histocompatibility complex (MHC) complexes requires their interaction with the resident endoplasmic reticulum (ER) chaperone calnexin, which for certain proteins functions as a lectin specific for monoglucosylated glycans. In the current report, we studied the expression of MHC class I proteins in BW wild type thymoma cells (BW WT) and glucosidase II-deficient BW PHAR2.7 cells. Consistent with a requirement for glucose (Glc) trimming for interaction of class I proteins with calnexin, we found that nascent H-2Kk proteins associated with calnexin in untreated BW WT cells, but not in BW WT cells treated with the glucosidase inhibitor castanospermine (cas), or in untreated glucosidase II-deficient BW PHAR2.7 cells. Surprisingly, we found that H-2Kk expression occurred with similar efficiency in BW PHAR2.7 cells as in BW WT cells and that formation of nascent H-2Kk complexes was perturbed by cas treatment in BW WT cells but not in BW PHAR2.7 cells. Finally, it was noted that expression of the molecular chaperone Bip was markedly increased in BW PHAR2.7 cells relative to BW WT cells, which is suggested to play a role in regulating the expression of H-2Kk complexes in BW PHAR2.7 cells. The current study demonstrates that Glc trimming is required for efficient interaction of nascent H-2Kk proteins with calnexin; that expression of MHC class I proteins can, under certain conditions, proceed effectively in the absence of Glc trimming and calnexin association; and that Bip expression is markedly increased under conditions where diglucosylated glycans persist on nascent glycoproteins within the ER. These data are consistent with the hypothesis that alternative oligomerization pathways exist for class I proteins within the quality control system of the ER that have differential requirements for removal of Glc residues from nascent glycan chains.

The ankle jerk and the tibial H-reflex: a clinical and electrophysiological correlation.

In an attempt to correlate the tibial H-reflex with the ankle jerk, we evaluated 130 reflexes in 65 patients (35 men and 30 women). All the ankle jerks were graded by one of the authors (BK) without the knowledge of the H-reflex results. The maximal H-reflex amplitude, and the maximal H/maximal M amplitudes correlated in a positive fashion with the ankle jerk (r = 0.75 and 0.69 respectively). In contrast, there was no correlation between the H-reflex latency and the ankle jerk (r = -0.11). We conclude that, in most situations, the tibial H-reflex amplitude and the ankle jerk correlate well; their discordance is infrequent (11.5%).

In vivo function of regulatory DNA sequence elements of a major histocompatibility complex class I gene.

Major histocompatibility complex class I genes are expressed in nearly all somatic tissues, although their level of expression varies. By analysis of a set of promoter deletion mutants introduced into transgenic mice, a complex regulatory element, consisting of overlapping enhancer and silencer activities, is demonstrated to function as a tissue-specific regulator of class I expression. The enhancer activity predominates in lymphoid tissues but not in nonlymphoid tissues. In contrast to the tissue-specific functions of the complex regulatory element, a second novel silencer element is shown to function in both lymphoid and nonlymphoid tissues. The complement of DNA-binding factors in different cell lines is shown to correlate with the levels of class I expression.

Magnetic brain stimulation and brain size: relevance to animal studies.

Magnetic brain stimulation (MBS) is widely used for the investigation of brain function in man, but there have been only a few reports of its safety in animals. These results were predominantly benign, but the effectiveness of stimulation in animals is unclear. Because the stimulators produced obvious motor effects in humans, or had a comparable peak magnetic field strength, they were assumed to produce comparable electric field intensities and neuronal effects in animal brains. We tested this assumption using 3 stimulus coils of different sizes and design, plus 6 saline-filled spheres that spanned a range of volume from 0.5 to 1800 ml. The induced electric field diminished monotonically with decreasing radius, by factors of 4.7-6.2 at the extremes of size. Comparable results were found using a mathematical model. These results suggest that the efficiency of magnetic stimulation is drastically reduced in smaller brains, and that threshold and safety studies in some animal models may not be valid.

Induction of transient neurological dysfunction in baboons by platelet microemboli.

BACKGROUND AND PURPOSE: To investigate experimental mechanisms of reversible cerebral dysfunction, we produced transient focal cerebral ischemia in five baboons by unilateral perfusion of internal carotid territories with platelet microemboli generated endogenously. METHODS: Platelet microemboli were formed by incorporating segments of Dacron vascular graft for 1 hour as unilateral carotid arterio-arterial shunts. Platelet embolization was assessed by ultrasonography and isotopic imaging; cerebral function was evaluated by measurements of somatosensory evoked potentials and clinical motor performance. RESULTS: Platelet microemboli, detected by transcranial Doppler ultrasonography, accumulated rapidly in the shunted carotid hemispheric territory. Indium-111-labeled platelets reached a maximum value of 3.2 +/- 0.8 x 10(9) platelets in the dependent hemisphere of five animals after 20 minutes of carotid blood flow through the grafts when measured in real time by continuous scintillation camera imaging. The retained 111In-platelet microemboli cleared from the cerebral vasculature within 1 hour after removing the grafts. Corresponding blood markers of in vivo thrombus formation, beta-thromboglobulin, platelet factor 4, and fibrinopeptide A, increased eightfold to 20-fold after incorporating graft segments and normalized within 1 hour after removing the grafts. Coincidently, focal neurological function was temporarily impaired, as shown by the ipsilateral loss of somatosensory evoked potentials 5 minutes after initiating platelet microembolization, with restoration 1 hour after removing the grafts in five baboons, and contralateral hemiparesis in two recovered baboons, with complete resolution by 24 hours. CONCLUSIONS: Endogenously generated platelet microemboli accumulate transiently in the dependent cerebral circulation and produce corresponding focal neurological dysfunction that resolves within hours after microembolization.

31P magnetic resonance spectroscopy suggests impaired mitochondrial function in AZT-treated HIV-infected patients.

Prompted by the report of a mitochondrial myopathy associated with chronic administration of zidovudine (AZT), an inhibitor of mitochondrial DNA synthesis, we obtained 31P magnetic resonance spectra from the calf muscles of AZT-treated patients and age-matched control subjects at rest and during an exercise protocol with a 12-second time resolution. The recovery of phosphocreatine following exercise reflects mitochondrial oxidative function and was significantly delayed in the AZT-treated patients (time constants, 43.3 +/- 12.5 seconds versus control subjects, 24.4 +/- 3.9 seconds). These findings support the hypothesis that the myopathy associated with chronic AZT results from the inhibitory effects of AZT on mitochondrial DNA synthesis and, secondarily, on the inhibition of mitochondrial oxidative metabolism.

A complex regulatory DNA element associated with a major histocompatibility complex class I gene consists of both a silencer and an enhancer.

A novel regulatory element which contributes to the regulation of quantitative, tissue-specific differences in gene expression has been found between -771 and -676 bp upstream of the major histocompatibility complex (MHC) class I gene, PD1. Molecular dissection of this element reveals the presence of two overlapping functional activities: an enhancer and a silencer. Distinct nuclear factors bind to the overlapping enhancer and silencer DNA sequence elements within the regulatory domain. The levels of factors binding the silencer DNA sequence in different cell types are inversely related to levels of class I expression; in contrast, factors binding the enhancer DNA sequence can be detected in all cells. In cultured cell lines, inhibition of protein synthesis leads to the rapid loss of silencer complexes, with a concomitant increase in both enhancer complexes and MHC class I RNA. From these data, we conclude that a labile silencer factor competes with a constitutively expressed, stable enhancer factor for overlapping DNA-binding sites; the relative abundance of the silencer factor contributes to establishing steady-state levels of MHC class I gene expression.

Striking similarities between the regulatory mechanisms governing yeast mating-type genes and mammalian major histocompatibility complex genes.

Expression of a mammalian major histocompatibility complex (MHC) class I gene is in part regulated by a silencer DNA sequence element which binds a complex of silencer factors. This negative regulatory system is shown to be strikingly similar to the yeast alpha 2 mating-type repression system. A moderate DNA sequence homology exists between the MHC class I silencer DNA element and the yeast alpha 2 operator. Mammalian silencer factors specifically bind to the yeast alpha 2 operator DNA and also specifically interact with a yeast alpha 2-binding protein. Furthermore, the alpha 2 operator functions as a silencer element in mammalian cells when placed upstream of a MHC class I promoter.