We have analyzed the production of tumor necrosis factor alpha (TNF-alpha) induced by in vitro infection with African swine fever (ASF) virus (ASFV) and the systemic and local release of this inflammatory cytokine upon in vivo infection. An early increase in TNF-alpha mRNA expression was detected in ASFV-infected alveolar macrophages, and high levels of TNF-alpha protein were detected by ELISA in culture supernatants from these cells. When animals were experimentally infected with a virulent isolate (E-75), enhanced TNF-alpha expression in mainly affected organs correlated with viral protein expression. Finally, elevated levels of TNF-alpha were detected in serum, corresponding to the onset of clinical signs. TNF-alpha has been reported to be critically involved in the pathogenesis of major clinical events in ASF, such as intravascular coagulation, tissue injury, apoptosis, and shock. In the present study, TNF-alpha containing supernatants from ASFV-infected cultures induced apoptosis in uninfected lymphocytes; this effect was partially abrogated by preincubation with an anti-TNF-alpha specific antibody. These results suggest a relevant role for TNF-alpha in the pathogenesis of ASF.

When integrated as a transgene in one or a few copies, the -90 35S promoter of cauliflower mosaic virus confers expression in roots with little or no expression in cotyledons and leaves. The responsible cis element, activation sequence-1 (as-1), can bind to the nuclear factor ASF-1 as well as to the transcription factor TGA1a. Here, we show that microinjection of 104 molecules of TGA1a per cotyledon cell activated transgenes containing as-1-linked promoters. Transgenes with promoters linked to the octopine synthase (ocs) element, which also binds TGA1a, responded similarly. The acidic, N-terminal segment of TGA1a is important for transcription activation in vivo because a deletion mutant without the first 80 amino acids was inactive. Finally, we show that the -90 35S-[beta]-glucuronidase (GUS) fusion gene conferred GUS expression in cotyledon cells when injected at 50,000 copies per cell. Collectively, these results provide support for the hypothesis that the undetectable expression of the as-1-linked transgene in cotyledon cells is most likely a result of its inability to compete for a limiting amount of its cognate transcription factor(s), presumably TGA1a or related proteins.

Daily observations were made on the excretion of African swine fever (ASF) virus by pigs infected intranasally or by contact. Two strains of virus having mean death times of approximately 3 and 6 days were used, the latter being recently isolated from a warthog.First excretion usually occurred by the nasopharyngeal route, as early as 1 or 2 days before the onset of fever in many cases. The titres of pharyngeal and nasal swabs rose rapidly to reach mean levels of about 10(4)-10(5) HAD 50 at 48-72 hr. following the onset of pyrexia. Virus in the secretions of the conjunctiva or lower urogenital tract appeared later and did not attain such high levels. Faecal and urinary excretion was of relatively little significance, except in slower infections caused by the recent warthog virus.These results are discussed in relation to the known failure of infected pigs to transmit the disease to stallmates during the first 12-24 hr. of pyrexia and also in relation to recent work on the pathogenesis of ASF in domestic swine.

Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was suggested previously (Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943-953). To identify these kinases, regulation of the binding of the nucleoplasmic region (NK, amino acid residues 1-211) of LBR to sperm chromatin was studied using a cell cycle-dependent Xenopus egg extract in vitro. The binding was stimulated on specific phosphorylation of the NK fragment by an S-phase egg extract. Protein depletion with beads bearing SF2/ASF, which binds SR protein kinases, abolished this stimulation, suggesting that an SR protein kinase(s) is responsible for the activation of LBR. This was confirmed by direct phosphorylation and activation with recombinant SR protein-specific kinase 1. The binding of the NK fragment to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor, roscovitine, and protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant NK fragment showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.

Digital breast tomosynthesis (DBT) is a three-dimensional (3D) x-ray imaging modality that reconstructs image slices parallel to the detector plane. Image acquisition is performed using a limited angular range (less than 50 degrees) and a limited number of projection views (less than 50 views). Due to incomplete data sampling, image artifacts are unavoidable in DBT. In this preliminary study, the image artifacts in DBT were investigated systematically using a linear system approximation. A cascaded linear system model of DBT was developed to calculate the 3D presampling modulation transfer function (MTF) with different image acquisition geometries and reconstruction filters using a filtered backprojection (FBP) algorithm. A thin, slanted tungsten (W) wire was used to measure the presampling MTF of the DBT system in the cross-sectional plane defined by the thickness (z-) and tube travel (x-) directions. The measurement was in excellent agreement with the calculation using the model. A small steel bead was used to calculate the artifact spread function (ASF) of the DBT system. The ASF was correlated with the convolution of the two-dimensional (2D) point spread function (PSF) of the system and the object function of the bead. The results showed that the cascaded linear system model can be used to predict the magnitude of image artifacts of small, high-contrast objects with different image acquisition geometry and reconstruction filters.

Curcumin (CM), a well-known dietary pigment derived from Curcuma longa L., possess anticancer activities against a variety of tumors including human breast carcinoma. In combination with docetaxel, CM has been used in breast cancer management in the clinic. In order to explore the possible mechanism of anticancer activity of CM, in the present study, we aimed to identify proteins involved in the anticancer activity of CM in human breast cancer cell line MCF-7 using the two-dimensional electrophoresis (2-DE)-based proteomic analysis. MCF-7 cells were cultured at 37°C in an atmosphere of 5.0% CO(2). All the following experiments were repeated three times. Cell viability assay showed that after a 48-h incubation CM dose-dependently inhibited cell growth with an IC(50) value of 47.42 μM. Treatment of CM at 47.42 μM for 48 h induced apoptosis as determined by nuclear morphologic changes of Hoechst stained cells and flow cytometric analysis of Annexin V-FITC/PI stained cells. Proteomic analysis identified 12 differentially expressed proteins which contributed to multiple functional activities such as DNA transcription, mRNA splicing and translation, amino acid synthesis, protein synthesis, folding and degradation, lipid metabolism, glycolysis, and cell motility. Among them 7 proteins were up-regulated and 5 down-regulated. The up-regulated ones were verified by quantitative real-time PCR. The down-regulated proteins, TDP-43, SF2/ASF and eIF3i, as well as up-regulated ones, 3-PGDH, ERP29, and platelet-activating factor acetylhydrolase IB subunit beta positively contribute to the anticancer activity of CM in MCF-7 cells. These molecules are implicated in the bioactivities of CM for the first time. The findings of this study would shed new insights for systematically understanding the mechanisms of CM in breast cancer intervention.

Protein-carbohydrate interactions are involved in diverse regulatory processes. To help understand the mechanics and kinetics of dissociation of receptor-ligand complexes, we have analyzed the separation of lactose and the N-glycan chains of asialofetuin (ASF) from three lectins and an immunoglobulin G fraction by surface plasmon resonance at zero force and by atomic force microscopy with variations of the external force. While the (AB)2 agglutinins from Ricinus communis (RCA) and Viscum album (VAA) show structural homology, the homodimeric galectin-1 from bovine heart (BHL) has no similarity to the two plant lectins except for sharing this monosaccharide specificity. The beta-galactoside-binding immunoglobulin G (IgG) fraction from human serum provides a further model system with distinct binding-site architecture. The k(off) constants for the two plant agglutinins were independent of the nature of the ligand at 1.1-1.3 x 10(-3) s(-1), whereas the geometry of ligand and binding site presentation affected this parameter for BHL (0.5 x 10(-3) s(-1) for lactose and 1 x 10(-3) s(-1) for ASF) and IgG (1.3 x 10(-3) s(-1) for lactose and 0.55 x 10(-3) s(-1) for ASF). When assessing comparatively the rupture forces at a loading rate of 3 nN/s with lactose as ligand, 34 +/- 6 pN (BHL), 36 +/- 4 pN (IgG), 47 +/- 7 pN (VAA), and 58 +/- 9 pN (RCA) were measured. For the same loading rate the rupture forces for the receptor-ASF interactions were found to be 37 +/- 3 pN (BHL), 43 +/- 5 pN (VAA), 45 +/- 6 pN (IgG), and 65 +/- 9 pN (RCA). The variation of the pulling velocity revealed in all cases a linear dependence between the rupture force and the natural logarithm of the loading rate. Performing probability density and Monte Carlo calculations, the potential barrier widths, which determine the inverse dynamic dependence with the rate of force elevation, increased from 4 A (RCA) and 7 A (VAA and IgG) to 10 A (BHL) for the receptor-lactose interactions. Presenting ASF as ligand potential widths of 4 A for RCA and IgG and 6 A for VAA and BHL were obtained. Since the dissociation kinetics at zero force apparently cannot predict the behavior in force-driven experiments, these results reveal new insights into biological functions. The dissociation kinetics under force helps to explain the difference in the toxic potency of VAA and RCA and points to a function of the galectin in cis-crosslinking and in transient trans-bridging.

Retroviruses use unspliced RNA as mRNA for expression of virion structural proteins and as genomic RNA; the full-length RNA often constitutes the majority of the viral RNA in an infected cell. Maintenance of this large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarcoma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Components of the splicing apparatus appear to be involved in splicing inhibition since binding of a number of splicing factors (snRNPs and SR proteins) and assembly of a large complex (NRS-C) in nuclear extracts correlate with NRS-mediated splicing inhibition. In determining the requirements for NRS complex assembly, we show that NRS-C assembly can be reconstituted by addition of total SR proteins to an S100 extract that lacks these factors. Of the purified SR proteins tested, SF2/ASF was functional in NRS-C assembly, whereas SC35 and SRp40 were not. The participation of snRNPs in NRS-C assembly was addressed by selectively depleting individual snRNPs with oligonucleotides and RNase H or by sequestering critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR proteins, but not U2 snRNP, are involved in NRS-C assembly.

METHODS

Prospective study.

OBJECTIVE

To prospectively evaluate sequential pulmonary function tests (PFTs) at a minimum 2-year follow-up after an open anterior spinal fusion (ASF) with instrumentation for thoracic AIS.

BACKGROUND

Anterior spinal fusion with instrumentation is currently undergoing evaluation as an alternative to posterior spinal fusion (PSF) for thoracic adolescent idiopathic scoliosis (AIS). However, the effect of an open thoracotomy on pulmonary function in these patients is unknown.

METHODS

Fifty-one patients with thoracic AIS with an average age of 15+0 (range 11+2 to 20+5) had PFTs consisting of volume (FVC), flow (FEV-1), and total lung capacity (TLC). Parameters were obtained preoperatively, and at 3 months, 1 year, and a minimum 2-year follow-up. All patients had a single or double open thoracotomy with the diaphragm kept intact. Fusion levels ranged from T4 (most proximal) to L1 (most distal). The average preoperative thoracic coronal Cobb measurement was 53 degrees (range 38 degrees to 80 degrees ), and the average postoperative coronal measurement was 24 degrees (range 7 degrees to 49 degrees ). The average preoperative thoracic sagittal kyphosis (T5-T12) averaged 22 degrees (range 10 degrees to 58 degrees ), and the average postoperative sagittal kyphosis measured 29 degrees (range 7 degrees to 67 degrees ).

RESULTS

There was a significant decline (P< or =0.05) in PFT absolute values (L) of 19%-FVC, 15%-FEV-1, and 11%-TLC at 3 months postoperatively with subsequent improvement and no statistical difference between preoperative and 2-year postoperative values. When evaluating percent predicted values, there was a statistical decline (P< or =0.05) at 3 months postoperatively averaging 19% FVC, 14% FEV-1, and 12% TLC. These values returned to within 94% to 96% of baseline by the 2-year follow-up visit, but were still statistically less than the preoperative values (P</=0.05).

CONCLUSIONS

Pulmonary function following thoracotomy with ASF with instrumentation demonstrated a significant decline of 3-month postoperative PFT values, but returned to preoperative baseline absolute values (L) by the 2-year follow-up visit. The percent predicted values returned to within 95% of baseline 2 years postoperatively. Scoliosis surgeons should be aware of these findings when deciding upon the approach (anterior versus posterior) utilized for thoracic AIS.

African swine fever (ASF) is one of the most severe diseases of pigs; it has a drastic impact on the pig industry, causing serious socio-economic consequences to pig farmers and pork producers. In Europe, there are currently two main clusters of infection; one in Sardinia caused by strains of African swine fever virus (ASFV) belonging to genotype I and another in Eastern Europe caused by strains of ASFV belonging to genotype II. The latter is inducing an acute form of ASF and it represents a serious threat to the pig sector. ASF is a disease for which there is no effective vaccine; therefore, prevention has a pivotal role in the control strategy of the disease. This review describes the main preventive measures to adopt to mitigate the risk of ASF spread in pig farming systems.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is caused by loss of the survival motor neuron gene, SMN1. SMA treatment strategies have focused on production of the SMN protein from the almost identical gene, SMN2. Valproic acid (VPA) is a histone deacetylase inhibitor that can increase SMN levels in some SMA cells or SMA patients through activation of SMN2 transcription or splicing correction of SMN2 exon 7. It remains to be clarified what concentration of VPA is required and by what mechanisms the SMN production from SMN2 is elicited. We observed that in two fibroblast cell lines from Japanese SMA patients, more than 1mM of VPA increased SMN2 expression at both the transcript and protein levels. VPA increased not only full-length (FL) transcript level but also exon 7-excluding (Δ7) transcript level in the cell lines and did not change the ratio of FL/Δ7, suggesting that SMN2 transcription was mainly activated. We also found that VPA modulated splicing factor expression: VPA increased the expression of splicing factor 2/alternative splicing factor (SF2/ASF) and decreased the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). In conclusion, more than 1mM of VPA activated SMN2 transcription and modulated the expression of splicing factors in our SMA fibroblast cell lines.

METHODS

Multicenter analysis of 2 groups of patients surgically treated for Lenke 5C adolescent idiopathic scoliosis (AIS).

OBJECTIVE

Compare patients with Lenke 5C scoliosis surgically treated with anterior spinal fusion with dual rod instrumentation and anterior column support with patients surgically treated with posterior release and pedicle screw instrumentation.

BACKGROUND

Treatment of single, structural, lumbar, and thoracolumbar curves in patients with AIS has been the subject of some debate. Advocates of the anterior approach assert that their technique spares posterior musculature and may save distal fusion levels, and that with dual rods and anterior column support the issues with nonunion and kyphosis have been obviated. Advocates of the posterior approach assert that with the change to posterior pedicle screw based instrumentation that correction and levels are equivalent, and the posterior approach avoids the issues with nonunion and kyphosis. This report directly compares the results of posterior versus anterior instrumented fusions in the operative treatment of adolescent idiopathic Lenke 5C curves.

METHODS

We analyzed 62 patients with Lenke 5C based on radiographic and clinical data at 2 institutions: 31 patients treated with posterior, pedicle-screw instrumented fusions at 1 institution (group PSF); and 31 patients with anterior, dual-rod instrumented fusions at another institution (group ASF). Multiple clinical and radiographic parameters were evaluated and compared.

RESULTS

The mean age, preoperative major curve magnitude, and preoperative lowest instrumented vertebral (LIV) tilt were similar in both groups (age: PSF = 15.5 years, ASF = 15.6 years; curve size: PSF = 50.3 degrees +/- 7.0 degrees , ASF = 49.0 degrees +/- 6.6 degrees ; LIV tilt: PSF = 27.5 degrees +/- 6.5 degrees , ASF = 27.8 degrees +/- 6.2 degrees ). After surgery, the major curve corrected to an average of 6.3 degrees +/- 3.2 degrees (87.6% +/- 5.8%) in the PSF group, compared with 12.1 degrees +/- 7.4 degrees (75.7% +/- 14.8%) in the ASF group (P < 0.01). At final follow-up, the major curve measured 8.0 degrees +/- 3.0 degrees (84.2% +/- 5.8% correction) in the PSF group, compared with 15.9 degrees +/- 9.0 degrees (66.6% +/- 17.9%) in the ASF group (P = 0.01). This represented a loss of correction of 1.7 degrees +/- 1.9 degrees (3.4% +/- 3.7%) in the PSF group, and 3.8 degrees +/- 4.2 degrees (9.4% +/- 10.7%) in the ASF group (P = 0.028). The LIV tilt decreased to 4.1 degrees +/- 3.4 degrees after surgery in the PSF group, and 4.5 degrees +/- 3.7 degrees in the ASF group. At final follow-up, the LIV tilt was 5.1 degrees +/- 3.5 degrees in the PSF group, and 4.5 degrees +/- 3.7 degrees in the ASF group. EBL was identical in both groups, and length of hospital stay was significantly (P < 0.01) shorter in the PSF group (4.8 vs. 6.1 days). There were no complications in either group which extended hospital stay or required an unplanned second surgery.

CONCLUSIONS

At a minimum of 2-year follow-up, adolescents with Lenke 5C curves demonstrated statistically significantly better curve correction, less loss of correction over time, and shorter hospital stays when treated with a posterior release with pedicle screw instrumented fusion compared with an anterior instrumented fusion with dual rods for similar patient populations.

Morphological data obtained by electron microscopy have shown that African swine fever virus adapted to VERO cells enters swine macrophages, its natural host cell, by a mechanism of receptor-mediated endocytosis. Binding studies with 3H-labeled virus and competition experiments with UV-inactivated virus have shown that the virus entry that leads to a productive infection in swine macrophages is mediated by saturable binding sites on the plasma membrane. The virus also penetrated into rabbit macrophages that do not produce infectious virus and initiated the synthesis of some early viral proteins; however, the viral replication cycle was aborted since viral DNA synthesis did not occur. The interaction of ASF virus particles with rabbit macrophages was mediated by nonsaturable binding sites, suggesting that the lack of specific receptors in these cells may be related to the absence of a productive infection. A similar abortive infection was detected in macrophages from other virus-resistant animal species.

African swine fever virus (ASFV) infection usually results in an acute haemorrhagic disease with a mortality rate approaching 100% in domestic pigs. However, pigs can survive infection with less-virulent isolates of ASFV and may become chronically infected. Surviving animals are resistant to challenge with homologous or, in some cases, closely related isolates of the virus indicating that pigs can develop protective immunity against ASFV. During asymptomatic, non-virulent ASFV infections natural killer cell activity increases in pigs, suggesting this cell type plays a role in ASFV immunity. Furthermore, depletion of CD8(+) lymphocytes from ASFV immune pigs demolishes protective immunity against related virulent viruses. This suggests that ASFV specific antibody alone is not sufficient for protection against ASFV infection and that there is an important role for the CD8(+) lymphocyte subset in ASFV protective immunity. These results were supported by DNA immunization studies, demonstrating a correlation between the protection afforded against lethal challenge and the detection of a large number of vaccine-induced antigen-specific CD8(+) T-cells. Peripheral blood mononuclear cells (PBMCs) from ASF immune pigs protected from clinical disease show higher proportions of ASFV specific CD4(+)CD8(high+) double positive cytotoxic T cells than PBMCs from ASF immune but clinically diseased pig. The frequency of ASFV specific IFNγ producing T cells induced by immunization correlates to the degree of protection from ASFV challenge, and this may prove to be a useful indicator of any potential cross-protection against heterologous ASFV isolates.

Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3'-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3'-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3'-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3'-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

The continuing circulation of African swine fever (ASF) in Russia and in the Trans-Caucasian countries has led to increased efforts in characterizing the epidemiology of ASF. For a better insight in epidemiology, quantitative data on virus excretion is required. Until now, excretion data has mainly focused on the initial stages of the disease. In our study we have studied ASF virus (ASFV) excretion dynamics in persistently infected animals. For this purpose, virus excretion through different routes was quantified over 70 days after infection. Three virus isolates of moderate virulence were used: the Brazil'78, the Malta'78 (a low and a high inoculation dose) and the Netherlands'86 isolate. For each isolate or dose, 10 animals were used. All (Brazil'78 group), or three animals per group were inoculated and the other animals served as contact animals. It was shown that dose (Malta'78 low or high) or infection route (inoculated or naturally infected) did not influence the ASFV excretion (p>0.05). Nasal, ocular and vaginal excretions showed the lowest ASFV titres. Virus was consistently present in the oropharyngeal swabs, showing two peaks, for up to 70 days. Virus was occasionally present in the faeces, occasionally with very high titres. Viral DNA persisted in blood for up to 70 days. The results presented in this study show that a high proportion of persistently infected animals shed virus into the environment for at least 70 days, representing a possible risk for transmission and that should be considered in future epidemiological analysis of ASF.

African Swine Fever (ASF) is an important contagious haemorrhagic viral disease affecting swine whose notification is mandatory due to its high mortality rates and the great sanitary and socioeconomic impact it has on international trade in animal and swine products. This disease only affects porcine species, both wild and domestic, and produces a variety of clinical signs such as fever and functional disorders of the digestive and respiratory systems. Lesions are mainly characterized by congestive-haemorrhagic alterations. ASF epidemiology varies significantly between countries, regions and continents, since it depends on the characteristics of the virus in circulation, the presence of wild hosts and reservoirs, environmental conditions and human social behaviour. Furthermore, a specific host will not necessarily always play the same active role in the spread and maintenance of ASF in a particular area. Currently, ASF is endemic in most sub-Saharan African countries where wild hosts and tick vectors (Ornithodoros) play an important role acting as biological reservoirs for the virus. In Europe, the disease has been endemic since 1978 on the island of Sardinia (Italy) and since 2007, when it was first reported in Georgia, in a number of Eastern European countries. It is also endemic in certain regions of the Russia Federation, where domestic pig and wild boar populations are widely affected. By contrast, in the affected eastern European Union (EU) countries where ASF is currently as epidemic, the on-going spread of the disease affects mainly wild boar populations located in restricted areas and, to a much less extent, domestic pigs. Unlike most livestock diseases, no vaccine or specific treatment is currently available for ASF. Therefore, disease control is mainly based on early detection and the application of strict sanitary and biosecurity measures. Epidemiology of ASF is very complex by the existence of different virus circulating, reservoirs and a number of scenarios, and the on-going spread of the disease through Africa and Europe. Survivor pigs can remain persistently infected for months which may contribute to virus transmission and thus the spread and maintenance of the disease, thereby complicating attempts to control it.

OBJECTIVE

As both habituation of pattern reversal visual evoked potentials (PR-VEP) (Schoenen J, Wang W, Albert A, Delwaide PJ. Potentiation instead of habituation characterizes visual evoked potentials in migraine patients between attacks. Eur J Neurol 1995;2:115-122) and intensity dependence of auditory evoked cortical potentials (IDAP) (Wang W, Timsit-Berthier M, Schoenen J. Intensity dependence of auditory evoked potentials in migraine: an indication of cortical potentiation and low serotonergic neurotransmission? Neurology 1996;46:1404-1409) were found abnormal in migraine between attacks, we have searched for intraindividual correlations between both tests in 59 migraine patients (22 with aura [MA], 37 without aura [MO]) and in 23 healthy volunteers (HV).

METHODS

Amplitude change of the PR-VEP N1-P1 was measured between the 1st and 5th block of 50 sequential averagings during continuous stimulation at 3.1 Hz. IDAP was computed from N1-P2 amplitudes of 100 averagings during stimulations at 40, 50, 60 and 70 dB SL. Amplitude-stimulus intensity function (ASF) slopes and amplitude changes between 40 and 70 dB were calculated. MO and MA differed from HV in PR-VEP amplitude change (P=0.007) and IDAP slope (P = 0.0004).

RESULTS

There was no significant correlation between VEP amplitude changes and IDAP slopes, nor between the latter two and attack frequency or disease duration. A negative correlation was found between the amplitude of the first block of averaged responses and potentiation of VEP in all subject groups (P = 0.03) as well as between the amplitude of the auditory evoked potential, at 40 dB, and the percentage of amplitude increase between 40 and 70 dB in MO (P = 0.004) and MA (P = 0.007). ASF slopes and 40 dB amplitudes were significantly correlated only in the MA group (P = 0.002). These results confirm the interictal deficit of habituation in cortical processing of repetitive visual and auditory information in migraine. Since there is no intraindividual correlation between the cortical responses to these sensory modalities they are complementary tools for the study of migraine and may help to identify subgroups of patients with distinct pathophysiological mechanisms.

CONCLUSIONS

The strong negative correlation between the initial amplitude of evoked potentials and their amplitude increase during subsequent averaging confirms that the response potentiation in migraine is likely to be due to a reduced preactivation level of sensory cortices.

RNA polymerase II, and specifically the C-terminal domain (CTD) of its largest subunit, has been demonstrated to play important roles in capping, splicing, and 3' processing of mRNA precursors. But how the CTD functions in these reactions, especially splicing, is not well understood. To address some of the basic questions concerning CTD function in splicing, we constructed and purified two fusion proteins, a protein in which the CTD is positioned at the C terminus of the splicing factor ASF/SF2 (ASF-CTD) and an RS domain deletion mutant protein (ASFDeltaRS-CTD). Significantly, compared to ASF/SF2, ASF-CTD increased the reaction rate during the early stages of splicing, detected as a 20- to 60-min decrease in splicing lag time depending on the pre-mRNA substrate. The increased splicing rate correlated with enhanced production of prespliceosomal complex A and the early spliceosomal complex B but, interestingly, not the very early ATP-independent complex E. Additional assays indicate that the RS domain and CTD perform distinct functions, as exemplified by our identification of an activity that cooperates only with the CTD. Dephosphorylated ASFDeltaRS-CTD and a glutathione S-transferase-CTD fusion protein were both inactive, suggesting that an RNA-targeting domain and CTD phosphorylation were necessary. Our results provide new insights into the mechanism by which the CTD functions in splicing.

Ticks of the Ornithodoros moubata complex were collected from domestic pig sties and dwelling houses, and from a warthog habitat, and tested for the presence of African swine fever (ASF) virus. Collections were made in 9 of the 24 districts of Malawi, these being primarily the districts in which O. moubata is most numerous. ASF virus was isolated from ticks collected in both domestic pig sties and houses in certain villages in Mchinji district where ASF outbreaks had recently occurred. Mchinji district is in the centre of a large ASF enzootic area which stretches into other districts of Malawi and also into Zambia and Mozambique. The high titre of virus in some of the ticks demonstrates that O. moubata can act as a virus reservoir and potential vector of disease in the field situation in Malawi.

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate expression of their target messenger RNAs. We recently demonstrated that primary miRNA transcripts (pri-miRNAs) retained at transcription sites are processed with enhanced efficiency, suggesting that pri-miRNA processing is coupled to transcription in mammalian cells. We also observed that transiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. Here, we show that pri-miRNAs containing a self-cleaving hepatitis delta ribozyme accumulate in the nucleoplasm after release from their transcription sites, but are not efficiently processed. Pri-miRNAs with ribozyme-generated 3' ends do not localize to SC35-containing foci, whereas cleaved and polyadenylated pri-miRNA transcripts with or without the pre-miRNA hairpin do. Pri-miRNA/SC35 foci contain a number of proteins normally associated with SC35 domains, including ASF/SF2, PABII, and the prolyl isomerase, Pin1. In contrast, RNA polymerase II and PM/Scl-100 do not strongly colocalize with pri-miRNAs in SC35-containing foci. These data argue that pri-miRNA/SC35-containing foci are not major sites of pri-miRNA processing and that pri-miRNA processing is coupled to transcription. We discuss the implications of our findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.

Alternative splicing in mammalian cells has been suggested to be largely controlled by combinatorial binding of basal splicing factors to pre-mRNA templates. This model predicts that distinct sets of pre-mRNA splicing factors are associated with alternatively spliced transcripts. However, no experimental evidence for differential recruitment of splicing factors to transcripts with distinct splicing fates is available. Here we have used quantitative single-cell imaging to test this key prediction in vivo. We show that distinct combinations of splicing factors are recruited to sites of alternatively spliced transcripts in intact cells. While a subset of serine/arginine protein splicing factors, including SF2/ASF, SC35, and SRp20, is efficiently recruited to the tau gene when exon 10 is included, these factors are less frequently associated with tau transcription sites when exon 10 is excluded. In contrast, the frequency of recruitment of several other splicing factors is independent of splicing outcome. Mutation analysis of SF2/ASF shows that both protein-protein as well as protein-RNA interactions are required for differential recruitment. The differential behavior of the various splicing factors provides the basis for combinatorial occupancy at pre-mRNAs. These observations represent the first in vivo evidence for differential association of pre-mRNA splicing factors with alternatively spliced transcripts. They confirm a key prediction of a stochastic model of alternative splicing, in which distinct combinatorial sets of generic pre-mRNA splicing factors contribute to splicing outcome.

Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively. We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors. To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies. We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor. The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments. We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them. Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction. Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection.

TGA1a and PG13 constitute a family of tobacco basic leucine zipper (bZIP) proteins that bind to activating sequence-1 (as-1), which is one of the multiple regulatory cis elements of the cauliflower mosaic virus (CaMV) 35S promoter. After truncation of the CaMV 35S promoter down to position -90 (CaMV 35S [-90] promoter), transcription stringently depends on the presence of as-1, which is recognized by nuclear DNA binding proteins called ASF-1. The role of the TGA1a/PG13 bZIP family in the formation of ASF-1 and in transcriptional activation of the CaMV 35S (-90) promoter has not yet been demonstrated in vivo. We constructed transgenic tobacco plants expressing a mutant of potato PG13, which lacks its wild-type DNA binding domain. This mutant acts as a trans-dominant inhibitor of ASF-1 formation and of expression from the CaMV 35S (-90) promoter, showing that PG13 can specifically interact with proteins necessary for these processes. Although we did not observe any other obvious phenotypic changes, these transgenic plants are a potentially valuable tool in identifying whether TGA1a and PG13 are involved in controlling promoters encoded in the plant genome.