The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein.

A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene. Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP). Its interaction with the 3' splice site occurs rapidly even at 0 degrees C, does not require adenosine triphosphate, and is altered by certain mutations in the 3' splice site region. Binding is surprisingly insensitive to treatment of the extract with micrococcal nuclease. The U5 particle is the only abundant Sm snRNP with a capped 5' end that is equally resistant to micrococcal nuclease. This suggests that, in addition to the U1 and U2 snRNP's, U5 snRNP's participate in pre-mRNA splicing.

Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro.

The neuron-specific N1 exon of the mouse c-src transcript is normally skipped in nonneuronal cells. In this study, we examined the sequence requirements for the exclusion of this exon in nonneuronal HeLa cell nuclear extracts. We found that the repression of the N1 exon is mediated by specific intron sequences that flank the N1 exon. Mutagenesis experiments identified conserved CUCUCU elements within these intron regions that are required for the repression of N1 splicing. The addition of an RNA competitor containing the upstream regulatory sequence to the HeLa extract induced splicing of the intron downstream of N1, indicating that the competitor sequence binds to splicing repressor proteins. The similarities between this mechanism for src splicing repression and the repression of other regulated exons point to a common role of exon-spanning interactions in splicing repression.

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.

A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon.

The mouse c-src gene contains a short neuron-specific exon, N1. To characterize the sequences that regulate N1 splicing, we used a heterologous gene, derived from the human beta-globin gene, containing a short internal exon that is usually skipped by the splicing machinery. Various fragments from the src gene were inserted into the globin substrate to measure their effects on the splicing of the test exon. These clones were transiently expressed in neuronal and nonneuronal cell lines, and the level of exon inclusion was measured by primer extension. Several sequences from the N1 exon region induced the splicing of the heterologous exon. The most powerful effect was seen with a sequence from the intron downstream of the N1 exon. This sequence acted as a strong splicing enhancer, activating splicing of the test exon when placed in the intron downstream. The enhancer was strongest in neuronal LA-N-5 cells but also activated splicing in nonneuronal HEK293 cells. Deletion and linker scanning mutagenesis indicate that the enhancer is made up of multiple smaller elements that must act in combination. One of these elements was identified as the sequence UGCAUG. Three copies of this element can strongly activate splicing of the test exon in LA-N-5 neuroblastoma cells. These component elements of the src splicing enhancer are also apparently involved in the splicing of other short cassette exons.

hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells.

The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.

Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein.

Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.

Alternative RNA splicing in the nervous system.

Tissue-specific alternative splicing profoundly effects animal physiology, development and disease, and this is nowhere more evident than in the nervous system. Alternative splicing is a versatile form of genetic control whereby a common pre-mRNA is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. In the nervous system, thousands of alternatively spliced mRNAs are translated into their protein counterparts where specific isoforms play roles in learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. The essential nature of this process is underscored by the finding that its misregulation is a common characteristic of human disease. This review highlights the current views of the biological phenomenon of alternative splicing, and describes evidence for its intricate underlying biochemical mechanisms. The roles of RNA binding proteins and their tissue-specific properties are discussed. Why does alternative splicing occur in cosmic proportions in the nervous system? How does it affect integrated cellular functions? How are region-specific, cell-specific and developmental differences in splicing directed? How are the control mechanisms that operate in the nervous system distinct from those of other tissues? Although there are many unanswered questions, substantial progress has been made in showing that alternative splicing is of major importance in generating proteomic diversity, and in modulating protein activities in a temporal and spatial manner. The relevance of alternative splicing to diseases of the nervous system is also discussed.

Differential regulation of progestin receptor immunoreactivity in the rabbit oviduct.

Cellular distribution of progestin receptor immunoreactivity (PR-IR) in paraffin-embedded rabbit oviduct tissue was determined using a monoclonal antibody and the indirect immunoperoxidase method. Ampullar and isthmic tissue from ovariectomized animals primed with 17 beta-estradiol or vehicle alone was examined. As expected, in the absence of estradiol, PR-IR was rarely observed in the ampulla. Estradiol treatment increased PR-IR in cell nuclei of the stroma and muscularis of the ampulla, but not of the epithelium. In contrast, PR-IR was seen in epithelial cell nuclei of the isthmus in the absence of estradiol. Surprisingly, while estradiol treatment increased PR-IR in cell nuclei of the stroma and muscularis of the isthmus, it decreased PR-IR in the epithelium. These results suggest for the first time a differential regulation of progestin receptors in the ampulla and isthmus of the oviduct. In particular, while estradiol treatment increased PR-IR in the stroma and muscularis and was without effect on PR-IR in the epithelium of the ampulla, it decreased PR-IR in the epithelium of the isthmus.

Cholestasis in transplant patients--what is the role of cyclosporin?

The use of the drug cyclosporin is limited by toxicity. It would be advantageous to develop therapeutic monitoring of cyclosporin which would predict the development of clinical toxicity. In the present study, alternative methods of measuring cyclosporin levels were evaluated in a heterogenous population of transplant patients, comparing a fluorescent polarization immunoassay using a non-specific polyclonal antibody, which measures both cyclosporin and its main metabolites, and a specific high-performance liquid chromatography assay for unchanged cyclosporin in blood. Neither measured variable alone correlated with laboratory evidence of renal toxicity (serum creatinine) or liver toxicity (serum glutamate transaminase, lactic dehydrogenase, alkaline phosphatase, or serum bilirubin). The relationship between metabolites and parent cyclosporin was quantitated using the ratio of cyclosporin levels determined by fluorescent polarization immunoassay over levels determined by high-performance liquid chromatography. A cohort of patients with markedly elevated ratios of cyclosporin were identified. When patients' data were reviewed collectively and individually there was a correlation between an elevated ratio and the serum bilirubin (r = 0.41, P less than 0.001). This association could be either due to cyclosporin as a cause of cholestasis or to cholestasis from any cause resulting in metabolite accumulation. Further studies are needed to clarify the role of cyclosporin in hepatic dysfunction and develop early, specific markers for this cyclosporin-associated toxicity.

Equality in survival of X and Y chromosome-bearing human spermatozoa.

Human X and Y chromosome-bearing spermatozoa survived equally well during washing and resuspension in buffers of pH 5.2 and 8.0 and during incubation in these buffers for 11 hours at 37 degrees C. This suggests that alteration of the ratio of living X- and Y-bearing spermatozoa by direct treatment is not an effective method of sex ratio alteration.

The effect of ion-exchange column chromatography on separation of X and Y chromosome-bearing human spermatozoa.

Separation of X and Y chromosome-bearing spermatozoa has been attempted using ion-exchange column chromatography, with cation- and anion-exchange resins of low, intermediate, and high ionic strength. Examination of F-bodies on the Y chromosome of treated human sperm and progeny resulting from insemination of treated rabbit spermatozoa indicates that in none of the cases investigated did the treatment cause a separation of X and Y chromosome-bearing spermatozoa. The treatment does appear to filter out dead rabbit (and bull) spermatozoa, but the possible beneficial effects of this phenomenon are as yet uninvestigated.

Treatment of persistent glaucoma secondary to periocular corticosteroids.

PURPOSE: To describe two patients with uveitis who developed increased intraocular pressure that was unresponsive to maximum medical therapy eight and 13 months after periocular injection of triamcinolone acetonide. METHODS: Excised periocular tissue was analyzed for corticosteroid activity by gas chromatography and mass spectrometry. RESULTS: Excision of the periocular tissue, which contained visible triamcinolone acetonide, resulted in a normal intraocular pressure within 14 days in both patients. Analysis of the excised tissue disclosed residual corticosteroid in one of the two patients. CONCLUSION: Removal of periocular tissue containing injected corticosteroids may facilitate the management of patients developing increased intraocular pressure unresponsive to maximum medical therapy.

The effects on rat pups when nitrofen (4-(2,4-dichlorophenoxy)nitrobenzene) was applied dermally to the dam during organogenesis.

Nitrofen (4-(2,4-dichlorophenoxy)nitrobenzene; TOK herbicide) was administered dermally as an aqueous dilution of an emulsifiable concentrate on Day 6-15 of gestation to pregnant Sprague-Dawley rats at dose levels of 0, 0.3, 0.6, 1.2 and 12.0 mg/kg/day. No maternal toxicity occurred. At 12 mg/kg neonatal survival was reduced and the animals that died had a high incidence of diaphragmatic hernias. Survivors showed increased incidences of diaphragmatic hernias and of missing or reduced Harderian glands, chromodarcryorrhea (a clear red exudate around the eyes), and a high frequency of slight to severe dilation of the renal pelvis. Thyroid and Harderian gland weights were significantly depressed in Day 42 survivors of both sexes at 12.0 mg/kg; liver and lung weights were decreased, and renal weight was increased in the females. At 12.0 mg/kg thyroid weights of males and females were significantly depressed at 146 days postnatal. The incidence of dilated kidneys was increased at 0.3 mg/kg and higher. No effect was observed at any dose level on time to eye opening, time to vaginal opening, mitotic index of the liver, or T3 levels in the dams or the offspring, and no gross behavioral effects were recorded. The no observable effect level was estimated to be 0.28 mg/kg in males and 0.17 mg/kg in females using a mathematical extrapolation.

The relation between newborn hemoglobin F fractions and risk factors for sudden infant death syndrome.

OBJECTIVES: The aims of this study were to determine and compare fetal hemoglobin (HbF) fractions at birth in newborns exposed and not exposed to selected factors that have been reported to increase the risk of sudden infant death syndrome (SIDS). Previous studies have implicated HbF in the etiology of SIDS by finding higher fractions in infants dying from SIDS compared to age-matched control infants. DESIGN: We performed a cross-sectional study using high-performance liquid chromatography to measure HbF fractions in newborn cord blood samples. Exposure to selected risk factors for SIDS was assessed through review of medical records. PARTICIPANTS: Six hundred thirty-three infants born at Via Christi Regional Medical Center-St Francis Campus, Wichita, Kan, from February 28 through August 5, 1997. MAIN OUTCOME MEASURE: Hemoglobin F fractions at birth were compared in newborns exposed and not exposed to selected risk factors associated with increased incidence of SIDS. RESULTS: Mean HbF fractions were significantly higher in preterm newborns of mothers who smoked and in term newborns with intrauterine growth restriction, pregnancy weight gain less than or equal to 9 kg, and pregnancy complications associated with reduced placental blood flow. An elevated newborn HbF fraction, defined as 77% or greater, was significantly associated with maternal smoking, maternal anemia, intrauterine growth restriction, and pregnancy complications associated with reduced placental blood flow. CONCLUSION: This study suggests a possible mechanism (HbF) by which previously identified factors may increase the risk of SIDS.

Relationship between progesterone suppression and pregnancy in rats.

Four luteolytic agents were administered to groups of pregnant rats to examine the quantitative relationship between serum progesterone levels and the maintenance of pregnancy. Each agent inhibited progesterone in a dose-dependent manner, however only three, azastene, thiosemicarbazone and dihydrotestosterone, adversely affected pregnancy. A statistical analysis of the data suggests that, regardless of the mechanism of action of a particular luteolytic agent, a treatment-induced depression of serum progesterone to concentrations less than 45% of that of the controls on day 11 of pregnancy is incompatible with pregnancy maintenance.

Urine cannabinoid analysis: an integrated multi-method approach.

The Enzyme Multiplied Immunoassay Technique (EMIT) Urine Cannabinoid Assay (Syva) was adapted for automated analysis on a centrifugal analyzer allowing for cost-effective, rapid, high-volume urine testing. Confirmation of positive EMIT results was accomplished by a modification of a recently published high performance liquid chromatographic (HPLC) technique for 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH). Further confirmation of the presence of THC-COOH was achieved by high efficiency thin layer chromatography (HETLC). The reported protocol was applied to urine samples obtained from an emergency toxicology population and a drug counseling population. Results indicated that specimens testing positive by all three methods suggest valid forensic evidence for the presence of THC-COOH.

Progesterone synthesis and histology of postpartum bovine corpora lutea.

In vitro progesterone (P(4)) synthesis by corpora lutea (CL) from the first, second or third ovulation after calving was compared and correlated with their histology and cytology. The CL were removed 7 to 12 days after ovulation, and luteal cells isolated by digestion with collagenase. The response of isolated cells to luteinizing hormone (LH) was determined. Hematoxylin-eosin stained tissues were used to study histology, and the distribution of cell types was estimated by stereological methods. Ovulation occurred within 25 days of calving and interovulatory intervals were short, 12.1 +/- 3.9 days and 12.6 +/- 4.8 days, respectively. The CL removed after first ovulation were smaller and contained fewer live cells than those obtained after subsequent ovulations. Stimulation by LH in vitro was independent of cycle number or day of cycle but was related to the histology of the tissue. The CL composed of large cells (> 24 mum) with vacuolated cytoplasm contained high amounts of P(4) but were not stimulated by LH. Conversely, CL composed of small and medium- sized cells (10 to 20 mum) and/or intact larger cells contained little P(4) but were stimulated by LH. These observations indicate that the response of postpartum CL to LH in vitro is dependent upon the structural integrity of the tissue at the time of removal. Furthermore, these observations suggest that the short life of CL during the postpartum period may not be due to the absence of luteotrophic support, but to the action of a luteolytic mechanism.

Intraoperative cortical somatosensory evoked potentials for detection of sciatic neuropathy during total hip arthroplasty.

Approximately 3% of patients undergoing hip arthroplasty develop postoperative sciatic neuropathy. The factors associated with changes in somatosensory evoked potentials (SSEP) and sciatic neuropathy were examined in patients undergoing hip arthroplasty, to evaluate whether the use of intraoperative SSEP could help reduce the incidence of postoperative sciatic neuropathy. Eighty-eight patients were assigned to either monitored or unmonitored groups. SSEP were recorded following peroneal nerve stimulation, using contralateral stimulation to detect systemic influences on SSEP. Amplitude reduction of less than 50% of control and/or latency increase of greater than 10% of control was considered significant, and surgical intervention was attempted to restore SSEP. Previous surgery and a lateral incision approach tended to be associated with sciatic neuropathy (p less than 0.053). The incidence of sciatic neuropathy in the monitored group (4.3%) was not different from the unmonitored group (2.4%). Isolated reduction in amplitude or prolongation in latency of the SSEP was not predictive of postoperative neurologic function of the sciatic nerve. Six patients, two of whom developed sciatic neuropathy, demonstrated complete flattening of the SSEP. Both of these patients had flattened SSEP for two or more surgical events (p less than 0.01) and flattened SSEP were present at the end of the surgical procedure. There were no false-negative SSEP changes. Simultaneous amplitude and latency changes appear to be predictive of sciatic nerve function following hip arthroplasty.