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Biomedical subjects

D L Black

Publications and source records attributed to D L Black.

At least 19 recordsLinked to original sources
PubMed1999

Mapping of the KHSRP gene to a region of conserved synteny on human chromosome 19p13.3 and mouse chromosome 17.

The K homology-type splicing regulatory protein, KSRP, activates splicing through intronic splicing enhancer sequences. It is highly expressed in neural cells and is required for the neural-specific splicing of the c-src N1 exon. In this study, we mapped the gene (gene symbols KHSRP and Khsrp) to human chromosome 19 by using radiation hybrid panels and to mouse chromosome 17 by studying an interspecific backcross panel. Human KHSRP is a positional candidate gene for familial febrile convulsion and Cayman type cerebellar ataxia. Comparative analysis of the human and mouse genomes indicates that the KHSRP gene is located in regions of conserved synteny between the two species.

Alternative Splicing↗
PubMed1999

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.

Alternative Splicing↗
PubMed1997

A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.

We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.

Amino Acid Sequence↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

A unique intronic splicing enhancer controls the inclusion of the agrin Y exon.

Alternative splicing of the agrin mRNA controls the ability of agrin protein to induce the clustering of acetylcholine receptors at the neuromuscular junction. Using a transfectable reporter gene, we show that one agrin alternative exon, the Y exon, is controlled by a regulatory sequence in the downstream intron. Portions of this intronic sequence have the properties of a splicing enhancer that can activate splicing of a heterologous exon when placed in the intron downstream. The regulatory region is complex in structure, containing several different elements capable of activating splicing. Individual enhancing elements differ in their cell-type specificity, and are not apparently synergistic, as two elements together induce lower splicing than either does separately. Essential nucleotides within these regulatory elements were identified by scanning mutagenesis across the active region. Interestingly, the elements do not appear similar to known intronic splicing enhancer elements. This Y exon enhancer and its components take part in an apparent combinatorial system of control where multiple regulatory elements of varying activity combine to produce a precisely cell-specific exon inclusion. As a major contributor to the regulation of the Y exon, the enhancer ultimately controls the properties of the agrin protein.

3T3 Cells↗
PubMed1996

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.

Anti-Inflammatory Agents↗
PubMed1995

The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event.

The proteins and RNA regulatory elements that control tissue-specific pre-mRNA splicing in mammalian cells are mostly unknown. In this study, a set of proteins is identified that binds to a splicing regulatory element downstream of the neuron specific c-src N1 exon. This complex of proteins bound specifically to a short RNA containing the regulatory sequence in neuronal extracts that splice the N1 exon. It was not seen in non-neuronal cell extracts that fail to splice this exon. UV-cross-linking experiments identified a neuron-specific 75-kD protein and several nontissue-specific proteins, including the 53-kD heterogeneous nuclear ribonucleoprotein F (hnRNP F), as components of this complex. Although present in both extracts, hnRNP F binds tightly to the RNA only in the neuronal extracts. A mutation in the regulatory RNA sequence, that inhibits N1 splicing in vivo, abolished formation of the neuron-specific complex and the binding of the neuron-specific 75-kD protein. Competition experiments in the two extracts show that the binding of the neuronal protein complex to the src pre-mRNA is required to activate N1 exon splicing in vitro. Antibody inhibition experiments indicate that the hnRNP F protein is a functional part of this complex. The assembly of regulatory complexes from both constitutive and specific proteins is likely to be a general feature of tissue-specific splicing regulation.

Animals↗
PubMed1995

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.

Alternative Splicing↗
PubMed1994

Efficacy of corticosomatosensory evoked potential monitoring in predicting and/or preventing sciatic nerve palsy during total hip arthroplasty.

Sciatic nerve palsy (SNP) is one of the most distressing complications associated with total hip arthroplasty. The authors used corticosomatosensory evoked potential monitoring in 290 consecutive patients in an attempt to predict when SNP might occur and to prevent its occurrence by changing the position of the retractors and/or the operated limb in response to intraoperative waveform changes. Despite these efforts there were 8 SNPs among the 290 patients (2.8%). The authors compared this group with 485 consecutive patients who were not monitored. In the latter group there were 13 SNPs (2.7%). There were two monitored patients (0.7%) with no intraoperative evidence of SNP who exhibited SNP after surgery (false negatives). Corticosomatosensory evoked potential monitoring was found to be neither effective in predicting SNP nor helpful in its prevention.

Evoked Potentials, Somatosensory↗
PubMed1992

Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro.

A conserved positive-acting RNA sequence was found to be required for the neuron-specific splicing of the mouse c-src N1 exon. The sequence lies in the intron between exons N1 and 4, close to the N1 donor site. Normally, only the neural-specific splicing of exon N1 required this sequence. When the intron downstream of N1 was shortened, splicing at the constitutive exon 4 acceptor also became dependent on the activating sequence. The neuronal and nonneuronal patterns of src splicing were reconstituted in vitro. HeLa cell extracts spliced exon 4 to exon 3, skipping exon N1. Weri-1 retinoblastoma cell extracts spliced exon 4 to exon N1 as well as to exon 3. Both patterns of splicing were dependent on the activating sequence. A 123 nt RNA containing just the activating sequence specifically inhibited both patterns of src splicing, indicating that factors bound to the activator were required for its effects.

Animals↗
PubMed1992

Role of maternal toxicity in assessing developmental toxicity in animals: a discussion.

The belief that any drug or chemical, when administered at a high enough dose, can be expected to produce fetal malformations is not consistent with the facts. However, the stress associated with maternally toxic doses can be expected to result in associated, often transient, fetal abnormalities that may not be the result of deviant organogenesis. Sometimes the toxicity toward the pregnant animal, including her embryos/fetuses since they are hardly in a sanctuary, is severe enough to result in resorption of the embryo or abortion of the fetus. Thus, it is possible that the embryolethality and other indications of developmental toxicity, produced by some drugs and chemicals, may be the result of a mechanism(s) other than selective toxicity toward the embryo. Also, some test materials have been shown to affect maternal homeostasis, thereby disrupting support to the embryo, without causing significant overt toxicity to the embryo or dam; e.g., the endocrine system of the dam is altered. Routine testing has thus far revealed a relatively limited number of true teratogens, although a large number of drugs and chemicals have resulted in fetal effects such as developmental variations when administered at doses that approach lethal levels. Such effects on the fetus should be expected when the maternal animals are stressed by the high dosages usually employed. A better understanding of the etiology and biological relevance of the embryo/fetal deviations often seen in developmental toxicology studies might help to avoid the sometimes unjustified withholding of potentially useful drugs and chemicals from the marketplace.

Abnormalities, Drug-Induced↗
PubMed1992

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.

Adolescent↗
PubMed1991

Reversal of bropirimine developmental toxicity with progesterone.

Bropirimine is an immunomodulator with experimental antiviral and antitumor activities. This pyrimidinone has been found to be embryolethal at doses (200 and 400 mg/kg) that produce only transient maternal toxicity, when administered to pregnant Upj:TUC(SD)spf rats on specific days of gestation. Serum analyses carried out in previous studies have shown marked decreases in progesterone levels in the 24 hr following bropirimine administration. In the present study, each of four groups of 5 bred rats and four groups of 10 bred rats was given bropirimine (gastric intubation) on Day 10 of gestation. Also, on Day 10 of gestation, progesterone (0.25, 0.50, or 1.00 mg/rat) was administered (im) twice (12-hr interval) a day to three of the groups of 5 dams each that had received bropirimine. In addition, three of the groups of 10 dams each received progesterone (0.25, 0.50, or 1.00 mg/rat) twice a day on Days 10-19 of gestation. Another group of 5 dams received progesterone only (0.50 mg/rat, b.i.d.) on Day 10 while a group of 10 dams received this same dose of progesterone on Days 10-19 of gestation. The groups containing 5 dams each were killed 24 hr postdosing while the groups containing 10 dams each were killed on Day 20 of gestation. The uteri were removed from the dams and examined. Administration of bropirimine alone resulted in the death of 100% of the embryos, at both the 24-hr and the Gestation Day 20 terminations. Exogenous administration of progesterone protected against bropirimine-mediated embryolethality; however, maternal effects were not alleviated. Thus, it appears likely that the embryolethality of bropirimine is the result of interruption of progesterone release from, or synthesis by, the corpora lutea, rather than direct toxicity toward the embryo, or lethal defects during organogenesis (terata).

Adjuvants, Immunologic↗