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R Reed

Publications and source records attributed to R Reed.

At least 37 records · Page 2Linked to original sources
PubMed1994

A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction.

We have isolated and determined the protein composition of the spliceosomal complex C. The pre-mRNA in this complex has undergone catalytic step I, but not step II, of the splicing reaction. We show that a novel set of 14 spliceosome-associated proteins (SAPs) and the essential splicing factor PSF are specifically associated with the C complex, implicating these proteins in catalytic step II. Significantly, immunodepletion and biochemical complementation studies demonstrate directly that PSF is essential for catalytic step II. Purified PSF is known to UV crosslink to pyrimidine tracts, and our data show that PSF UV crosslinks to pre-mRNA in purified C complex. Thus, PSF may replace the 3' splice site binding factor U2AF65 which is destabilized during spliceosome assembly. Finally, we show that SAPs 60 and 90, which are present in both the B and C complexes, are specifically associated with U4 and U6 snRNPs, and thus may have important roles in the functioning of these snRNPs during the splicing reaction.

Base Sequence↗
PubMed1994

Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114.

Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.

Amino Acid Sequence↗
PubMed1994

Occupational asthma in a latex doll manufacturing plant.

BACKGROUND: Occupational asthma caused by latex has been reported in health care workers and workers in glove manufacturing plants. OBJECTIVE: We report occupational asthma from latex in a newly identified occupational setting, a latex doll manufacturing plant. METHODS: We evaluated an index case of asthma associated with work in a latex doll manufacturing plant by performing a workplace challenge and evaluating the work environment. We then performed an occupational survey and skin testing of 22 workers in the doll manufacturing plant. RESULTS: The patient, a 21-year-old woman, had severe immediate bronchospasm within minutes of beginning a workplace challenge where sanding of latex parts was performed. Two of 22 workers surveyed (including the patient) reported flushing, rhinoconjunctivitis, and wheezing on exposure to sanded doll parts. These two workers were the only subjects surveyed to have a history of atopy and positive immediate-type skin test responses to a raw latex extract and to common aeroallergens. CONCLUSIONS: Sanding or grinding of solid latex during the manufacturing process may result in a significant incidence of occupational asthma and rhinoconjunctivitis from latex sensitization. Atopic workers appear to be most susceptible to developing latex sensitivity in this setting.

Adult↗
PubMed1994

Analysis of cerbB2 expression using a panel of 6 commercially available antibodies.

Results are presented of a study comparing cerbB2 (neu or Her2) expression as assessed immunohistochemically in breast neoplasia using a panel of 6 commercially available antibodies. The antibodies were examined utilizing conventional formalin fixed paraffin embedded tissue, and compared with molecular analysis of gene amplification. The aim was to determine the practical utility of each antibody, assessing ease of use, specific and non-specific staining characteristics, and expense, thus allowing a specific recommendation as to antibody of choice for immunohistochemical assessment of cerbB2 expression. Reassuringly, amongst the 38 breast lesions (36 carcinomas, 2 fibroadenomas) subjected to immunohistochemically (IHC) with the panel of 6 antibodies (Ab), no gross discrepancy of staining pattern was seen. Of the 38 cases, 10 were positive (26%), where at least one Ab demonstrated clear cytoplasmic membrane staining. Of a total of 45 breast lesions (43 carcinomas, 2 fibroadenomas), including all those examined by IHC, the total number of cases showing cerbB2 amplification by DNA analysis was 14 (31%). Using the DNA amplification as a base line for comparison, one Ab (No. 4) was found to stain 6 of the 14 cases of breast carcinoma that were assessed as showing amplification at the DNA level. Four Abs (1,3,5,6) stained 5 of these cases. However, Abs 3,4 and 6 displayed artefactual cytoplasmic staining (in the absence of membrane staining) that precluded the practical use of these reagents. Therefore, based on additional considerations of cost and ease of use, Ab No. 1 was finally chosen for recommendation from the 6 Ab panel.

Adult↗
PubMed1994

Patterns of speech disorders in schizophrenia and mania.

Formal thought disorder (FTD), defined as abnormal speech, has been associated with schizophrenia and likened to fluent aphasia. Whether FTD differentiates subtypes of schizophrenics and discriminates schizophrenics from other patients is unclear. We studied this issue by analyzing ratings of FTD of 170 schizophrenics and 62 manics. Eighty percent of emotionally blunted schizophrenics had FTD compared with 6.5% of manics. Factor analysis revealed verbiage disturbance and disorganized speech factors (44% of the variance). We assessed the discriminating ability of these factors, and compared these results to those from factors derived from Andreasen's positive/negative FTD construct, and to factors derived from speech and language diagnostic criteria for schizophrenia and mania in the proposed DSM-IV. Overall classifications were similar (91%, 91%, and 88%, respectively). We also found that FTD was related to emotional blunting, but not to other psychopathology.

Adolescent↗
PubMed1994

SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex.

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.

Base Sequence↗
PubMed1994

Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly.

Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF65, which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF65 and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.

Animals↗
PubMed1994

Integrin-mediated adhesive properties of uroepithelial cells are inhibited by treatment with bacterial toxins.

Gram-negative bacteria are a dominant cause of urinary tract infection, and their ability to produce toxins is an important virulence attribute. Cellular mechanisms triggered by the production of toxins in the lower urinary tract have not been completely defined. Ureteral epithelial cells (UT; A. Elgavish, Infect. Immun. 61: 3304-3312, 1993) have served as an in vitro model to explore the possibility that bacterial toxins act on UT by affecting integrin-mediated adhesive properties. The effect of treatment with lipopolysaccharides (LPS) from three strains of the gram-negative Escherichia coli [055:B5 (LPS-1), 0111:B4 (LPS-4), and 0127:B8 (LPS-5)] and lipoteichoic acids from two gram-positive bacteria, Streptococcus faecalis (LT-2) and Bacillus subtilis (LT-3), were examined. LPS-5 inhibited markedly UT attachment to collagen and fibronectin. LPS-4 had no effect, whereas LPS-1 inhibited UT attachment to collagen but not to fibronectin. The fact that LPS-5 and LT-2 inhibited an Arg-Gly-Asp sequence-sensitive component of UT attachment to fibronectin is consistent with the possibility that these toxins acted via a mechanism involving typical fibronectin receptors. UT spreading was inhibited markedly by LPS-1, LT-2, and LT-3, whereas LPS-4 and LPS-5 had no effect. Because clustering of integrins is a crucial step in integrin-mediated signal transduction, the possibility that toxins inhibited spreading by affecting clustering was tested. Treatment with LT-2, which inhibited spreading dramatically, abolished completely a UT cell population containing more than five to eight beta 1- or beta 4-subunit-containing integrin clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Correspondence between a mammalian spliceosome component and an essential yeast splicing factor.

None of the mammalian splicing factors that have been cloned corresponds to the yeast pre-messenger RNA splicing factors, the PRP proteins. Here, a generalizable strategy was used to isolate a complementary DNA encoding the mammalian spliceosome-associated protein (SAP) SAP 62. It is demonstrated that SAP 62 is the likely functional homolog of the yeast PRP11 protein. Both PRP11 and SAP 62 associate stably with the spliceosome, contain a single zinc finger, and display significant amino acid sequence similarity. Unlike PRP11, SAP 62 contains 22 proline-rich heptapeptide repeats at the carboxyl-terminus.

Amino Acid Sequence↗
PubMed1993

A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals.

The earliest detectable mammalian prespliceosome complex (E) contains the non-snRNP splicing factor U2AF, U1 snRNP, and several spliceosome-associated proteins (SAPs). We show that specific complexes, designated E3' and E5', assemble independently on RNAs containing only a 3' or 5' splice site, respectively. U2AF is enriched in E3', whereas U1 snRNP is enriched in E5'. Using a highly sensitive substrate-competition assay, we show that both the 5' splice site and the pyrimidine tract at the 3' splice site are required for efficient E complex assembly on intact pre-mRNA. We conclude that the 5' and 3' splice sites are associated functionally as early as E complex by either direct or indirect interactions between U1 snRNP and U2AF. Our observations predict that E complex assembly is a major control point for establishing splice site selection in both constitutively and alternatively spliced pre-mRNAs.

Animals↗
PubMed1993

Limitation of medical care: an ethnographic analysis.

This ethnographic study has shown how one attempt to apply ethical principles through a routine procedure failed to fit the clinical context and, in the two cases studied, served to counteract the very foundation these principles were based on--that patients or their families have the right to determine life-and-death decisions regarding code status. The results suggest that the use of well-meaning forms that are intended to facilitate decision making can, in the absence of appropriate guidelines, routinize the doctor-patient discourse to meet bureaucratic needs, narrowing rather than expanding understanding and communication. Bioethical principles implemented in abstraction, apart from the complex intricacies of the doctor-patient-family relationship and the sociocultural influences upon which this relationship is dependent, may be counter-productive to patient interests. As bioethicists and clinicians work to implement the demands of the Patient Self-Determination Act, they will undoubtedly try to forestall legal problems, assure ethical consistency, facilitate auditing, and promote documentation by creating forms. They may look to create inventories, such as the Limitation of Medical Care form described here, or turn to other, less explicit, means of documentation. This study suggests that, in these efforts, genuine attention should be given to patient concerns, not just to the ethical or institutional needs of medicine. This shift in focus from outcome to process can enhance patient and clinician satisfaction, help resolve difficulties in reaching consensus between involved decision makers, and return the power in DNR decision making to patients and families.

Adult↗
PubMed1992

Photodynamic therapy of human ovarian epithelial carcinoma, OVCAR-3, heterotransplanted in the nude mouse.

OBJECTIVE: Our objective was to develop an animal model for the study of photodynamic therapy in the treatment of human ovarian epithelial carcinoma. STUDY DESIGN: The human ovarian carcinoma OVCAR-3 was heterotransplanted into nude, athymic mice and treated with photodynamic therapy consisting of the hematoporphyrin derivative Photofrin II 10 ng/kg and argon-pumped dye laser light at 630 nm (200 J/cm2). Growth of tumors on one side of seven mice (treated tumors, n = 7) was compared with contralateral tumors (control tumors, n = 8) not exposed to laser. Hematoporphyrin derivative uptake was determined in tumor and other tissues. RESULTS: Photodynamically treated tumors were completely ablated, and all remained absent. Hematoporphyrin derivative uptake was nonselective for tumor compared with other tissues. CONCLUSION: This model provides reproducible parameters for the study of photodynamic therapy in the treatment of gynecologic malignancies and demonstrates the need for methods to increase selective uptake of hematoporphyrin derivatives.

Animals↗
PubMed1992

Intraperitoneal therapy of malignant ascites associated with carcinoma of ovary and breast using radioiodinated monoclonal antibody 2G3.

A phase I/II study of intraperitoneal (ip) radioimmunotherapy was conducted in ovarian or breast cancer patients with symptomatic chemotherapy-resistant ascites using a novel anti-mucin monoclonal antibody (mAb) 2G3 labeled with 131I. Tracer doses of 2 mCi [131I]2G3 were given by ip injection to 11 patients, followed by increasing therapeutic doses up to 150 mCi (cumulative) in 9 patients. There was no serious toxicity. Temporary palliation of ascites was observed in 3 of 4 patients who received doses greater than 50 mCi. Total body elimination half-life of the radiolabeled antibody assessed by gamma scintigraphy ranged from 95 to 250 hr, longer than data previously reported in patients without ascites treated with ip administered radiolabeled antibodies. However, uptake of radiolabel by tumor nodules was small and variable (2 x 10(-4) - 2 x 10(-2) % ID/g), and preferential uptake by tumor compared to normal peritoneum was observed in only 2 of 5 patients in whom biopsies were obtained. These results suggest that the observed palliation of ascites is due to prolonged retention of radiolabeled antibody in the peritoneal cavity even in the absence of specific targeting.

Adult↗
PubMed1992

Protein components specifically associated with prespliceosome and spliceosome complexes.

We have carried out a systematic analysis of the protein composition of highly purified mammalian spliceosomes. We show that > 30 distinct proteins, including 20 previously unidentified components [designated spliceosome-associated proteins (SAPs)], are specifically associated with the spliceosome in a salt-resistant complex. In contrast to these spliceosome-specific proteins, we show that hnRNP proteins are not tightly associated with purified prespliceosome and spliceosome complexes. The splicing factor U2AF65, U1 snRNP-specific proteins, and several SAPs are present in the earliest prespliceosome complex (E). A set of 10 proteins is then added to the first ATP-dependent prespliceosome complex (A), and concomitantly, a significant decrease in the level of U2AF65 is observed. The fully assembled spliceosome is formed by the addition of 12 proteins in a reaction that requires ATP and both the 5' and 3' splice sites.

Adenosine Triphosphate↗