Pigs in the United States were exposed to African swine fever (ASF) virus isolated from pigs in Brazil and the Dominican Republic. The former were examined for clinical response, lesions, viremia, and antibody response. Sequential blood samples were tested for the presence of ASF virus by the hemadsorption test (in swine buffy coat cell culture) and for antibody to ASF virus by the enzyme-linked immunosorbent assay. The incubation period was 3 to 5 days; inoculated pigs had fever for 8 to 16 days (mean 12.5 days) and viremia at 3 to 35 days after inoculation and few died. Inoculated pigs developed antibodies at 7 days after inoculation which were detectable until the termination of the experiment (10th month). Reinoculation of some of the surviving pigs with the homologous isolate at approximately 6 months after exposure did not induce clinical response, viremia, nor anamnestic antibody response. In contrast, challenge exposures of convalescent pigs with the Lisbon-60 viral strain approximately 5 weeks after exposure to the Brazilian strain produced death, in spite of an anamnestic antibody response.

African swine fever (ASF) is an infectious disease that causes heavy mortality in domestic pigs. At present there is no vaccine against ASF, and eradication in countries where the disease is endemic is based only on competent diagnosis programs and the sacrifice of infected animals. Due to the presence of natural attenuated strains, certain infection conditions may result in reduced mortality. In these situations, the disease can be diagnosed by detection of specific antibodies. The use of classical and validated diagnosis assays, such as ELISA and Indirect Immunofluorescence or Immunoblotting, allowed the eradication of ASF in the Iberian Peninsula in the 1990s. However, given that conventional tests include the use of antigens obtained from ASF virus (ASFV)-infected cells, they have several disadvantages, such as difficulties to achieve standardization and also the risks associated with the manipulation of live virus. Such drawbacks have led to the development of alternative and more robust systems for the production of ASFV antigens for use in anti-ASFV antibody detection systems. In the present review, we provide an update on current knowledge about antigen targets for ASFV serodiagnosis, the significant progress made in recombinant antigen production, and the refinement of ASF serological diagnostic assays. Moreover, we describe the accuracy of an ELISA developed for the serodiagnosis of ASFV in Africa. This assay is based on a novel p30 recombinant protein (p30r) obtained from an Eastern African viral isolate (Morara strain), which shares 100% amino acid sequence identity with the Georgia virus isolate. That study included the analyses of 587 field sera collected from domestic pigs and warthogs in Senegal (West Africa), the Democratic Republic of Congo (Central Africa), Mozambique (South-East Africa), and South Africa. The results revealed that the novel p30r-based ELISA allows the accurate detection of antibodies against ASFV, independently of the geographical origin of the sera.

The cloned organic anion transporters from rat, mouse, and winter flounder (rOAT1, mOAT1, fROAT) mediate the coupled exchange of alpha-ketoglutarate with multiple organic anions, including p-aminohippurate (PAH). We have isolated two novel gene products from human kidney which bear significant homology to the known OATs and belong to the amphiphilic solute facilitator (ASF) family. The cDNAs, hOAT1 and hOAT3, encode for 550- and 568-amino-acid residue proteins, respectively. hOAT1 and hOAT3 mRNAs are expressed strongly in kidney and weakly in brain. Both genes map to chromosome 11 region q11.7. PAH uptake by Xenopus laevis oocytes injected with hOAT1 mRNA is increased 100-fold compared to water-injected oocytes. PAH uptake is chloride dependent and is not further increased by preincubation of oocytes in 5 mM glutarate. Uptake of PAH is inhibited by probenicid, alpha-ketoglutarate, bumetanide, furosemide, and losartan, but not by salicylate, urate, choline, amilioride, and hydrochlorothiazide.

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of swine which can be transmitted through direct contact with infected animals and their excretions or indirect contact with contaminated fomites. The shedding of ASFV by infected pigs and the stability of ASFV in the environment will determine the extent of environmental contamination. The recent outbreaks of ASF in Europe make it essential to develop disease transmission models in order to design effective control strategies to prevent further spread of ASF. In this study, we assessed the shedding and stability of ASFV in faeces, urine and oral fluid from pigs infected with the Georgia 2007/1 ASFV isolate. The half-life of infectious ASFV in faeces was found to range from 0.65 days when stored at 4°C to 0.29 days when stored at 37°C, while in urine it was found to range from 2.19 days (4°C) to 0.41 days (37°C). Based on these half-lives and the estimated dose required for infection, faeces and urine would be estimated to remain infectious for 8.48 and 15.33 days at 4°C and 3.71 and 2.88 days at 37°C, respectively. The half-life of ASFV DNA was 8 to 9 days in faeces and 2 to 3 days in oral fluid at all temperatures. In urine, the half-life of ASFV DNA was found to be 32.54 days at 4°C decreasing to 19.48 days at 37°C. These results indicate that ASFV in excretions may be an important route of ASFV transmission.

METHODS

A prospective study.

OBJECTIVE

Comparison study of radiologic and clinical outcomes, efficiency, and cost between anterior spinal fusion (ASF) and posterior spine fusion (PSF) in surgical treatment of moderate lumbar/thoracolumbar adolescent idiopathic scoliosis (AIS).

BACKGROUND

ASF and PSF indicated for lumbar and thoracolumbar adolescent idiopathic scoliosis surgical treatment have respective advantages and disadvantages. However, up until today, a related prospective AIS comparative study has rarely been reported.

METHODS

Thirty-two cases in this prospective study with patients enrolled in either method A or B alternately in a sequence were divided into 2 groups. Group A underwent ASF with single solid rod and single screw constructs, and group B underwent PSF with segmental total pedicle screw system. Inclusion criteria were: (1) AIS diagnosis; (2) diagnosis classification as Lenke5CN type; (3) Cobb angles 35 degrees-60 degrees on anteroposterior view radiographs. Exclusion criteria were: (1) a history of spinal surgery; (2) age younger than 10 years; (3) Risser sign 0 degree; (4) lumbar/thoracolumbar kyphosis. All patients were observed with 2-year minimum follow-up (24-46 months). Clinical and radiologic outcomes of both groups A and B were analyzed.

RESULTS

Statistical t test or Mann-Whitney U test demonstrated no significant difference in preoperative age (P = 0.380), Risser sign (P = 0.733), magnitude (P = 0.936), flexibility (P = 0.815), apical vertebra rotation (AVR, P = 0.756), and apical vertebra translation (AVT, P = 0.355) of the lumbar/thoracolumbar curves, trunk shift (TS, P = 0.448), sagittal kyphosis from T5-T12 (P = 0.792) and sagittal lordosis from L1-L5 (P = 0.299). Average coronal correction of thoracolumbar/lumbar curves was 83% after surgery and 77% at follow-up in group A and 87% after surgery and 82% at follow-up in group B (P = 0.236 and P = 0.138). No significant differences were observed regarding correction of sagittal alignment, TS, AVT, AVR and hospitalization days on last follow-up between both groups (P>> 0.05). No pseudarthrosis, reoperation, neurologic complications, infection, and no other problems were observed. Excellent clinical fusion results were present in all patients on their last follow-up. However, significant differences were evident in group A in regards to reduced operative time (P = 0.046), reduced estimated blood loss (P = 0.003), decreased blood transfusion (P = 0.006), reduced implants cost and hospitalization expenses (P = 0.000). Additionally, group A had shorter fusion levels than group B (p50 = 4 vs. p50 = 5, P = 0.003).

CONCLUSIONS

ASF versus PSF comparison in treating moderate lumbar/thoracolumbar AIS did not show significant differences in regards to safety or efficacy but demonstrated shorter fusion levels, reduced surgical trauma and costs in ASF.

Critical protein-protein interactions among pre-mRNA splicing factors determine splicing efficiency and specificity. The serine/arginine proteins, a family of factors characterized by the presence of an RNA recognition motif and an arginine/serine domain, are essential for constitutive splicing and required for some alternative splicing decisions. ASF/SF2, SC35, and other members of the serine/arginine family, interact with the 70k protein of the U1 small nuclear ribonucleoprotein. The binding of this protein with ASF/SF2 is thought to enhance recognition of the 5' splice site of pre-mRNAs by the U1 small nuclear ribonucleoprotein. It has been clearly documented that the arginine/serine domain of ASF/SF2 is responsible for binding to the U1 70k protein. In this manuscript we characterize the segment in the human U1 70k protein that is both necessary and sufficient for ASF/SF2 binding. A domain within this segment, which begins with Arg240 and ends with Asp270, was shown to bind specifically to the arginine/serine domain of ASF/SF2 using a yeast two-hybrid system and a far Western assay. Mutational analysis of this segment suggested that several arginines are critical for the interaction with ASF/SF2 and for phosphorylation by SRPK1. Inspection of the sequence of the Arg248 to Asp270 region suggested this as an arginine/serine-like domain in U1 70k protein, and the data presented in this manuscript strongly support this view. Inspection of the human U1 70k protein sequence, comparison with homologues in other animal species, and mutational analysis indicated the importance of the sequence Arg-Arg-Arg-Ser-Arg-Ser-Arg-Asp, which is found repeated twice in the region from Arg248 to Asp270 in the human protein.

Transcription and pre-mRNA splicing are coordinated temporally and spatially, and both processes can influence each other. In particular, control of transcriptional elongation by RNA polymerase II has proved to be important for alternative splicing regulation. In this report we demonstrate that the efficiency of exon recognition by the splicing machinery is crucial for the elongation control. Alternative splicing of the fibronectin extra domain I (EDI) is because the polypyrimidine tract of its 3'-splice site occurs suboptimal. By mutating the polypyrimidine tract of EDI in two different positions, individually or in combination, and by disrupting its exonic splicing silencer, we managed to generate minigenes with increasing degrees of exon recognition. Improvement of exon recognition is evidenced by independence from the splicing regulator SF2/ASF for inclusion. The mutated minigenes were used to transfect human cells in culture and study the responsiveness of EDI alternative splicing to activation or inhibition of pol II elongation. Our results revealed that responsiveness of exon skipping to elongation is inversely proportional to 3'-splice site strength, which means that the better the alternative exon is recognized by the splicing machinery, the less its degree of inclusion is affected by transcriptional elongation.

The authors prepared water-soluble (WSF), urea-soluble (USF), alkali-soluble (ASF), sonicated (SF), sonicated insoluble (SIF) and membrane (MF) fractions of lens proteins from human senile and diabetic cataractous lenses and age-matched clear lenses. Levels of advanced glycation end products (AGEs) including carboxymethyl lysine (CML), a glycoxidation product, were determined by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA). Distribution of AGEs in the various protein fractions was ascertained by SDS-PAGE and Western blotting. An overall increase in the levels of AGEs in diabetic cataractous lenses as compared to senile cataractous lenses and clear lenses has been observed. ASF and SF, both of which originated from the urea-insoluble fraction, showed the highest levels of AGEs. However, no clear-cut differences in CML levels were seen among clear lenses and senile and diabetic cataractous lenses. AGEs were found to be distributed mostly in the high molecular aggregates in all the fractions. These data suggest that AGEs contribute to protein aggregation and subsequent insolubilization.

Previous observational studies in developing countries have suggested that diet quality, particularly increased animal source food (ASF) consumption, is positively associated with child cognitive development. This report presents findings from a study in rural Kenya, designed to test the impact of three different diets on the cognitive development of school children. Twelve schools with a total of 555 Standard 1 children (equivalent to U.S. Grade 1) were randomized to one of four feeding interventions: Meat, Milk, Energy or Control (no feeding). Feeding continued for seven school terms (21 mo), and cognitive tests were administered before the commencement of feeding and during every other term of feeding. Hierarchical linear random effects models and associated methods were used to examine the effects of treatment group on changes in cognitive performance over time. Analyses revealed that children receiving supplemental food with meat significantly outperformed all other children on the Raven's Progressive Matrices. Children supplemented with meat, and children supplemented with energy, outperformed children in the Control group on tests of arithmetic ability. There were no group differences on tests of verbal comprehension. Results suggest that supplementation with animal source food has positive effects on Kenyan children's cognitive performance. However, these effects are not equivalent across all domains of cognitive functioning, nor did different forms of animal source foods produce the same beneficial effects. Implications of these findings for supplementation programs in developing countries are discussed.

T cells from patients with systemic lupus erythematosus (SLE) produce insufficient amounts of the vital cytokine IL-2. We previously showed that SLE T cells express decreased levels of the T-cell receptor-CD3ζ chain and forced expression of CD3ζ into SLE T cells restores IL-2 production. We recently showed that the serine arginine protein splicing factor 2/alternative splicing factor (SF2/ASF) enhances the expression of CD3ζ chain by limiting the production of an unstable splice variant. Here we demonstrate that SF2/ASF levels are decreased in patients with SLE and more so in those with active disease. More importantly, we reveal a function of SF2/ASF, independent of T-cell receptor/CD3 signaling, whereby it is recruited to the IL-2 promoter, increases transcriptional activity, and enhances IL-2 production in SLE T cells. Our results demonstrate that SF2/ASF regulates IL-2 production and that decreased SF2/ASF expression in SLE T cells contributes to deficient IL-2 production.

Since 2007, African swine fever (ASF) has posed a serious threat to the European swine industry. In Poland, the numbers of reported outbreaks in pigs and affected areas grow every year. In 2018, the disease was noted in Western Europe, in Belgium specifically, where several hundred infected wild boars have been detected so far. In 2018, the virus unexpectedly emerged in pig holdings in eastern China, northern Mongolia, Vietnam, and Cambodia, causing worldwide concern about its further spread. Since there is still no vaccine available, the only approach to control the disease is biosecurity. Identification of potential sources of the virus is extremely important in light of its phenomenal survivability. The review summarises the current knowledge about ASFV survivability and resistance to environmental conditions, and discusses the role of indirect contact in spreading the disease.

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(®) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas.

METHODS

Retrospective review of a prospectively collected, multicentered database from the Scoliosis Research Society.

OBJECTIVE

To evaluate incidences of complications in a series of spinal fusions for Scheuermann kyphosis (SK) and to assess whether the incidence of complications is associated with patient age and surgical approach.

BACKGROUND

Although there is some evidence that adolescents have lower complication rates for spinal deformity surgery, this has not been well-documented for SK. Moreover, there is a lack of consensus on surgical approach for the management of SK.

METHODS

The Scoliosis Research Society morbidity and mortality database was queried to identify cases of SK from 2001 to 2004. Complications rates were analyzed based on patient age and surgical approach. Pediatric and adult patients were defined as <or=19 and >19 year old, respectively.

RESULTS

A total of 683 procedures involving spinal fusion for SK were identified. Mean patient age was 21 years (range: 5-75 years), with the majority (73%) of patients <or=19 years old. Procedures included 338 (49%) posterior spinal fusions (PSF), 73 (11%) anterior spinal fusions (<em>ASF</em>), and 272 (40%) same-day <em>ASF</em> and PSF. Ninety-nine complications were reported (14%). The most common complication was wound infection (3.8%). The acute neurologic complication rate was 1.9%, including 4 spinal cord injuries (0.6%). The mortality rate was 0.6%. Complications were more common among adult (22%) compared with pediatric patients (12%) (P = 0.002). The overall incidence of complications did not differ significantly between the PSF (14.8%) and same-day <em>ASF</em>/PSF (16.9%) procedures (P = 0.5).

CONCLUSIONS

The incidence of complications associated with spinal fusion for SK in adults is significantly greater than in pediatric patients. There were no significant differences in complication rates between PSF and same-day ASF/PSF procedures. These data may be used to counsel patients regarding complications associated with spinal fusion for SK in the hands of experienced spinal deformity surgeons.

The effects of human alternative splicing factor, ASF, on in vitro splicing of adenovirus E1A pre-mRNA were examined. E1A pre-mRNA is a complex substrate, and splicing in HeLa cell nuclear extracts produces six different RNAs using three alternative 5' splice sites and two 3' splice sites. Addition of excess ASF to splicing reactions produced a simplified splicing pattern, in which only one spliced product, 13S RNA, was detected. Inhibition of 12S and 9S splicing, which use 5' splice sites upstream of the 13S 5' splice site, extends previous observations that when multiple 5' splice sites compete for the same 3' splice site, ASF causes preferential selection of the proximal 5' splice site. However, inhibition of the other splices, which use a different upstream 3' splice site, represents a novel activity of ASF, as competition between 5' splice sites is not involved. The effect of ASF on 12S splicing was found to depend on its position relative to competing 5' splice sites, indicating that the ability of ASF to activate proximal 5' splice sites is position- but not sequence-dependent. Finally, addition of small amounts of ASF to ASF-lacking S100 extract was able to activate distal as well as proximal 5' splice sites in two of three pre-mRNAs tested, indicating that in these cases changes in the concentration of ASF alone can be sufficient to modulate alternative 5' splice site selection.

Protein 4.1R is a vital component of the red blood cell membrane cytoskeleton. Promotion of cytoskeletal junctional complex stability requires an erythroid differentiation stage-specific splicing switch promoting inclusion of exon 16 within the spectrin/actin binding domain. We showed earlier that an intricate combination of positive and negative RNA elements controls exon 16 splicing. In this report, we further identified 3 putative exonic splicing enhancers within exon 16 and investigated the function of the sequence CAGACAT in the regulation of exon 16 splicing. Mutation of these sequences leads to increased exclusion of exon 16 in both in vivo and in vitro splicing assays, indicating that CAGACAT is a functional exonic splicing enhancer. UV cross-linking further detects an approximately 33-kDa protein that specifically binds to the CAGACAT-containing transcript. An anti-SF2/ASF antibody specifically immunoprecipitates the approximately 33-kDa protein. Furthermore, SF2/ASF stimulates exon 16 inclusion in both in vitro complementation assays and minigene-transfected mouse erythroleukemia cells (MELCs). Finally, SF2/ASF expression is up-regulated and correlates with exon 16 inclusion in differentiated MELCs. These results suggest that increased splicing factor 2/alternative splicing factor (SF2/ASF) expression in differentiated mouse erythroleukemia mediates a differentiation stage-specific exon 16 splicing switch through its interaction with the exonic splicing enhancer.

OBJECTIVE

Shunt obstruction represents a permanent threat for patients with shunts, and its prevention and treatment are important parts of neurosurgeons' duties. Although there is little discussion regarding the need to reoperate for treatment of symptomatic shunt failure (SSF), the need to reoperate for treatment of asymptomatic shunt failure (ASF) is debated, as are the guidelines for the follow-up monitoring of patients with shunts. The goal of this study was to assess the effects of systematic follow-up monitoring and shunt revision for ASF; we reviewed our database to compare the results of shunt revision for ASF versus SSF.

METHODS

We defined ASF as shunt failure diagnosed for an asymptomatic patient during a systematic consultation. In our institution, children who receive shunts for treatment of hydrocephalus are systematically monitored in an outpatient clinic, with clinical examinations and plain x-rays. Among 1,564 children with shunts, who were monitored for a mean of 10.7 years, 1106 (70.7%) required at least one shunt revision. The indication for the first revision was SSF in 609 cases and ASF in 305 cases; the indication was not specified in 192 cases. We studied the surgical outcomes after the first shunt revision and compared the results for SSF and ASF.

RESULTS

After the first revision, shunt infections and subsequent shunt failure were significantly less frequent in the ASF group, compared with the SSF group. The interval between the first shunt revision and subsequent shunt failure was significantly longer in the ASF group.

CONCLUSIONS

Our data support the practice of systematic follow-up monitoring for patients with shunts, for the early diagnosis and systematic treatment of ASF.

The rat beta-tropomyosin gene encodes two tissue-specific isoforms that contain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus binding site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentially represent exonic splicing enhancers (ESEs). In this report we carried out substitution mutagenesis of these elements and show that some of them play a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular factors that binds to these motifs. Furthermore, we show that substrates that have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing. Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' splice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the polyuridine tract in the 3' splice site or consensus sequence in the 5' splice site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and consensus splice site sequences around exon 6 were present in the same pre-mRNA. These results support and extend the exon-definition hypothesis and demonstrate that sequences at the 3' splice site can facilitate use of a downstream 5' splice site. In addition, the data support the hypothesis that ESEs can compensate for weak splice sites, such as those found in alternatively spliced exons, thereby providing a target for regulation.

METHODS

A retrospective study was conducted.

OBJECTIVE

To compare the long-term outcomes after laminoplasty and anterior spinal fusion (ASF) for cervical myelopathy secondary to disc herniation.

BACKGROUND

There have been no reports of long-term comparative studies of laminoplasty and ASF for cervical myelopathy due to disc herniation.

METHODS

Of 21 patients who underwent ASF only between 1984 and 1987, 15 were followed up. Of 22 patients who underwent laminoplasty only between 1987 and 1994, 18 were followed up. There were no significant differences in preoperative prognostic factors between the 2 groups. Average follow-up was 15 years in the ASF group and 10 years in the laminoplasty group. Neurologic and radiologic results were examined.

RESULTS

Laminoplasty and ASF provided equal neurologic improvement. In the ASF group, additional surgery was required for bone graft complications in 2 patients and for adjacent spondylosis in 1. In the laminoplasty group, one patient had C5 palsy, and intractable axial pain developed in 5 patients after surgery, but no patients needed additional surgery.

CONCLUSIONS

Because the 2 procedures provided the same neurologic improvement, the risks of bone graft complication with ASF must be weighed against the risks of chronic neck pain associated with laminoplasty for determining the best technique. Therefore, because our present surgical strategy for cervical myelopathy due to disc herniation, laminoplasty is the procedure of choice except for a patient with single level disc herniation without developmental canal stenosis, who is considered to be a good candidate for ASF.

The SR proteins constitute a family of nuclear phosphoproteins which are required for constitutive splicing and also influence alternative splicing regulation. They have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain, rich in arginine and serine residues. The functional role of the different domains of SR proteins in constitutive splicing activity has been extensively studied in vitro; however, their contribution to alternative splicing specificity in vivo has not been clearly established. We sought to address how the modular domains of SR proteins contribute to alternative splicing specificity. The activity of a series of chimeric proteins consisting of domain swaps between different SR proteins showed that splice site selection is determined by the nature of the RRMs and that RRM2 of SF2/ASF has a dominant role and can confer specificity to a heterologous protein. In contrast, the identity of the RS domain is not important, as the RS domains are functionally interchangeable. The contribution of the RRMs to alternative splicing specificity in vivo suggests that sequence-specific RNA binding by SR proteins is required for this activity.

We have previously described human (HsSWAP) and mouse (MmSWAP) homologs to the Drosophila alternative splicing regulator suppressor-of-white-apricot (su(wa) or DmSWAP). DmSWAP was formally defined as an alternative splicing regulator by studies showing that it autoregulates splicing of its own pre-mRNA. We report here that mammalian SWAP regulates its own splicing, and also the splicing of fibronectin and CD45. Using an in vivo system of cell transfection, mammalian SWAP regulated 5' splice site selection in splicing of its own second intron. SWAP enhanced splicing to the distal 5' splice site, whereas the SR protein ASF/SF2 enhanced splicing to the proximal site. SWAP also regulated alternative splicing of the fibronectin IIICS region by promoting exclusion of the entire IIICS region. In contrast, ASF/SF2 stimulated inclusion of the entire IIICS region. Finally, SWAP regulated splicing of CD45 exon 4, promoting exclusion of this exon, an effect also seen with ASF/SF2. Experiments using SWAP deletion mutants showed that splicing regulation of the fibronectin IIICS region and CD45 exon 4 requires a region including a carboxyl-terminal arginine/serine (R/S)-rich motif. Since R/S motifs of various splicing proteins have been shown to interact with each other, these results suggest that the R/S motif in SWAP may regulate splicing, at least in part, through interactions with other R/S containing splicing factors.

METHODS

Retrospective clinical, radiographic, and patient outcome review of surgically treated adolescent idiopathic scoliosis.

OBJECTIVE

To evaluate the spontaneous correction of the noninstrumented proximal thoracic (PT) curve after isolated correction of the main thoracic (MT) curve by either an anterior (ASF) or posterior (PSF) instrumentation and fusion.

BACKGROUND

There are no studies comparing the structural PT curve response after anterior versus posterior instrumented fusion of the MT curve in adolescent idiopathic scoliosis.

METHODS

Eighty-five patients (single surgeon) with adolescent idiopathic scoliosis underwent operative instrumentation and fusion of their MT curve. All patients had a PT curve>> or =20 degrees (average 29 degrees, range 20-49 degrees; average residual side-bending 18 degrees, range 3-42 degrees ) and were evaluated for preoperative PT curve flexibility and postoperative curve correction after PSF with the PT curve not instrumented (n = 44) and ASF with the PT curve not instrumented (n = 41). Minimum follow-up was 2 years (average, 3.6 years). Preoperative, 1 week postoperative, and latest follow-up (minimum 2-year) full-length radiographs were evaluated for the PT, MT, and thoracolumbar-lumbar coronal, side-bending, and sagittal Cobb measurements, as well as T1 tilt, clavicle angle, radiographic shoulder height, and the PT, MT, and thoracolumbar-lumbar apical vertical translation. A patient outcome questionnaire was also completed to correlate patient satisfaction with respect to their shoulder balance and overall appearance.

RESULTS

The two groups were found to be statistically equivalent (P = 0.66) in terms of preoperative PT curve, MT curve, and MT side-bending curves, with the PT side benders slightly more flexible for the ASF (43%) versus the PSF group (31%) (P = 0.02). RADIOGRAPHIC: The spontaneous improvement in the PT curve was significant (P < 0.0001) in both groups. Additionally, this correction was maintained over time. However, the spontaneous PT curve correction was significantly greater after an ASF versus PSF correction of the MT curve on both the immediate postoperative (P =0.017) and minimum 2-year (P = 0.0024) evaluations, whereas the MT curve correction was the same in both groups (P = 0.45). There was no difference in the postoperative sagittal change in the PT curve (P = 0.12) between the two groups, and there was no difference in radiographic shoulder height (P = 0.5883). PATIENT OUTCOME: Both groups reported improvement in shoulder balance and clinical appearance, but there was no statistical difference between the two groups (P = 0.24). Additionally, no patients reported deterioration in either parameter.

CONCLUSIONS

Spontaneous proximal thoracic curve correction consistently occurs after instrumented correction of the main thoracic curve. Furthermore, this spontaneous correction is as good as or slightly better after an ASF versus PSF of the MT curve. The preoperative side bender radiographs (PT curve flexibility) positively correlate with the postoperative spontaneous PT curve correction.

Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae.

We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction.

The cis-located DNA sequence as-1 (Activation Sequence-1) from CaMV 35S promoter has been previously identified as an element that can confer inducibility by salicylic acid (SA) with immediate early kinetics. This sequence specifically binds to ASF-1 (Activation Sequence Factor-1), previously characterized in tobacco nuclear extracts. To assess whether modulation of ASF-1 binding activity can explain the activation of the as-1 sequence observed in vivo, we performed electrophoretic mobility shift assays using nuclear extracts from SA-treated and water-treated tobacco plants. Our results indicate that treatment of plants with SA increases ASF-1 binding to as-1 and to ocs, an as-1-like element from the Agrobacterium octopine synthase gene. In contrast, SA treatment has no effect on the binding of GT-1 factor to its target light-inducible box II element. Furthermore, treatment of nuclear extracts from SA-treated plants with alkaline phosphatase decreases ASF-1 binding to the as-1 element. This can be reversed by pretreatment with 10 mM NaF. Accordingly, pretreatment of nuclear extracts from control water-treated plants with ATP produces an increase in ASF-1 binding activity similar to that observed with SA. This effect of ATP is reversed by treatment with alkaline phosphatase and prevented by quercetin, a casein kinase II inhibitor. These results support the hypothesis that a nuclear protein kinase is involved in the immediate early events of transcriptional activation triggered by SA.