Water and sanitation for disasters.
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Community Participation↗
Biomedical subjects
R Reed
Publications and source records attributed to R Reed.
Protein composition of mammalian spliceosomes assembled in vitro.
This paper reports an analysis of the protein composition of highly purified mammalian spliceosomes isolated by a two-step large-scale affinity chromatography procedure. Splicing complexes were assembled in vitro on biotinylated pre-mRNA, fractionated by gel filtration, and then affinity-purified by binding to avidin-agarose. The purified spliceosomes are unexpectedly complex, containing at least 50 proteins that range in molecular mass from less than 14 to 200 kDa. Three complexes that assemble in the absence of ATP were also purified and characterized. These include a complex enriched in the small nuclear ribonucleoprotein particle U1 and non-specific complexes assembled either on pre-mRNA or an RNA lacking splice sites. Comparison between these complexes and the spliceosome revealed a distinct set of pre-mRNA-specific proteins and a set of proteins that bind to pre-mRNA only in the presence of ATP. Proteins in these two classes, many of which do not correspond in size to known small nuclear ribonucleoprotein particle proteins, are strong candidates for functional splicing components.
Animals↗
The organization of 3' splice-site sequences in mammalian introns.
A model pre-mRNA substrate was used to carry out a detailed investigation of the functional organization of sequences at the 3' end of mammalian introns. This analysis revealed a difference in the sequence requirements for the first and second steps of the splicing reaction (lariat formation and exon ligation, respectively). Maximal efficiencies of lariat formation require a pyrimidine stretch directly adjacent to the branch site. In addition, efficient lariat formation can be specified in at least two distinct ways, one that requires the AG dinucleotide at the 3' splice junction, and the other that does not: If the pyrimidine stretch is short (14 nucleotides), an adjacent AG is essential; in contrast, the AG is not required in the presence of a long pyrimidine stretch (26 nucleotides). In a pre-mRNA containing a long pyrimidine stretch, efficient lariat formation is observed when the branch site is located greater than 100 nucleotides upstream from the AG or when the AG is preceded by a purine stretch. Although splicing usually takes place at the first AG downstream from the branch site, both distance and sequence play roles in the efficiency of this reaction.
Animals↗
An electron microscopy and x-ray study of actin. I. Electron microscope. 1947.
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Actins↗
Thermal skin injury: I. Acute hemodynamic effects of fluid resuscitation with lactated Ringer's, plasma, and hypertonic saline (2,400 mosmol/l) in the rat.
Heart rate (HR), central venous pressure (CVP), mean arterial pressure (MAP), and cardiac index (CI) were measured in anesthetized rats subjected to a 40% body surface area full-thickness scald burn. Postburn intravenous fluid therapy with lactated Ringer's (5 ml/hr), plasma (2.5 ml/hr), or very hypertonic saline (2,400 mosmol/l) (0.75 ml/hr) was compared to unburned or burned, untreated controls. HR and CVP were not influenced significantly by thermal injury. MAP decreased steadily in the untreated group from 110 mmHg to 80 mmHg at 3 hr postburn. In the fluid-treated groups MAP did not change significantly. During the first 15 min postburn, CI was reduced to 58-71% of control values (P less than 0.01). CI increased during Ringer's and plasma infusion to 74-80% of control values (P less than 0.02 vs. unburned). Despite infusion therapy, hematocrit increased from 48 to 52%, clearly less than in the unresuscitated group (increase from 48 to 58%). Theoretically, the 2,400 mosmol/l saline would expand extracellular volume by five to six times the infused volume. Still, CI was reduced by 55% at 3 hr postburn in the hypertonic saline as well as in the burned, untreated group (P less than 0.001 vs. unburned). The low CI was mainly due to a reduced stroke volume.
Animals↗
Thermal skin injury: II. Effects on edema formation and albumin extravasation of fluid resuscitation with lactated Ringer's, plasma, and hypertonic saline (2,400 mosmol/l) in the rat.
Pentobarbital anesthetized rats were subjected to a 40% body surface area full-thickness scald burn. Intravenous fluid therapy was given as lactated Ringer's (5 ml/hr), plasma (2.5 ml/hr), or very hypertonic saline (2,400 mosmol/l) (0.75 ml/hr) and compared to unburned or burned, untreated controls. At 3 hr postburn, skin water and albumin content and extravasation of radiolabelled albumin were determined. Water content in injured skin increased by 35-78% (least in the untreated group, most in the plasma group) compared to unburned controls (P less than 0.05). After lactated Ringer's therapy water content increased even in unburned skin and in muscle (P less than 0.05). Tissue albumin mass increased generally slightly more than the increase in water content, from 37% (lactated Ringer's group) to 126% (plasma group) in burned areas. Extravasation rate of radiolabelled albumin increased 5-80 times in burned areas, most following plasma treatment (equivalent to 0.6-1.0 ml plasma/g dry weight/180 min). A major part of the estimated total fluid loss following therapy by lactated Ringer's took place in noninjured tissue. Plasma therapy gave less fluid accumulation in unburned tissues but more edema in the injured areas than lactated Ringer's.
Albumins↗
Purification and visualization of native spliceosomes.
Mammalian spliceosomes were purified in preparative amounts by gel filtration chromatography and shown to be functional by in vitro complementation experiments. The column fractions containing spliceosomes are enriched in the snRNAs U1, U2, U4, U5, and U6 and a subset of proteins present in the nuclear extract. Splicing intermediates, the entire set of snRNAs, and the enriched proteins can be immunoprecipitated with three different monoclonal antibodies that recognize snRNP determinants. At least one U1 snRNP is present in each spliceosome since the particles are quantitatively immunoprecipitated by an anti-U1 snRNP monoclonal antibody. Examination of the spliceosome fractions by EM revealed a relatively homogeneous population of 40-60 nm particles with a striking morphology. Evidence that these particles are spliceosomes is their sensitivity to micrococcal nuclease, their ATP-dependent assembly, and their immunoprecipitation with a trimethyl cap monoclonal antibody. In addition, pre-mRNA was visualized in the particles by EM.
Adenosine Triphosphate↗
Characterization of a cloned cDNA encoding rabbit myelin P2 protein.
Myelin P2 is a 14,800-Da cytosolic protein found in rabbit sciatic nerves. It belongs to a family of fatty acid binding proteins and shows a 72% amino acid sequence similarity to aP2/422, the adipocyte lipid binding protein, a 58% sequence similarity to rat heart fatty acid binding protein, and a 40% sequence similarity to cellular retinoic acid binding protein. In order to isolate cDNA clones representing P2, a cDNA library was constructed from poly(A+) RNA isolated from sciatic nerves of 10-day-old rabbit pups. By use of a mixed synthetic oligonucleotide probe based on the rabbit P2 amino sequence, 12 cDNA clones were selected from about 25,000 recombinants. Four of these were further characterized. They contained an open reading frame, which when translated, agreed at 128 out of 131 residues with the known rabbit P2 amino acid sequence. These cDNAs recognize a 1.9-kilobase mRNA present in sciatic nerve, spinal cord, and brain, but not present in liver or heart. The levels of P2 mRNA parallel myelin formation in sciatic nerve and spinal cord with maximal amounts being detected at about 15 postnatal days. This initial study will allow characterization of the P2 gene and its regulation, as well as further studies into the role of P2, the first metabolically active myelin-specific protein to be characterized at the genetic level.
Amino Acid Sequence↗
Exogenous ochronosis in the United States.
Two middle-aged black women reported hyperpigmentation of the face after using bleaching creams containing hydroquinone. Both noted initial lightening of facial pigmentation followed by progressive darkening of the areas to which the cream was applied. After biopsy, they were found to have exogenous ochronosis. These are the fifth and sixth cases of exogenous ochronosis reported in the United States. In this article we review the literature and discuss the possible pathogenesis of exogenous ochronosis.
Adult↗
Identification of a functional mammalian spliceosome containing unspliced pre-mRNA.
Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5' cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.
Animals↗
The role of the mammalian branchpoint sequence in pre-mRNA splicing.
We show that base substitutions in the mammalian branchpoint sequence (BPS) YNCUGAC dramatically reduce the efficiency of pre-mRNA splicing in vitro and alter 3' splice-site selection in vivo. Contrary to current dogma that an adenine residue at the appropriate distance from the 3' splice site is the primary determinant of lariat formation, we find that many mutations in the BPS virtually abolish splicing even though the position of this adenine is unchanged. Comparison of the analogous single-base changes in the mammalian and yeast BPSs revealed similar relative effects on splicing efficiency. However, in contrast to yeast, mammalian branchpoint mutations that decrease splicing efficiency severely do not prevent spliceosome assembly. Thus, mutations in the mammalian BPS appear to uncouple spliceosome assembly from cleavage at the 5' splice site and lariat formation.
Adenine↗
Family history of alcoholism does not predict neuropsychological performance in alcoholics.
We examined the relationship of history of alcoholism in first-degree relatives to neuropsychological performance of alcoholics abstinent from several weeks to several years. Eighty-four men were assigned to four groups based on "strength" of family history of alcoholism. The groups were: (1) "strong history," a parent plus another first-degree relative positive; (2) "moderate," parent only positive; (3) "weak," nonparent first-degree relative only positive; and (4) "negative," no first-degree relative positive. There were no significant between-group differences in NP performance. In other analyses there were no NP differences between alcoholics classified positive or negative purely on basis of paternal alcoholism, and no differences between subjects who had multigenerational versus unigenerational versus negative familial histories of alcoholism. It is concluded that genetic loading for alcoholism does not significantly affect the NP status of abstinent alcoholic groups equated for education, drinking history, and medical risk.
Adult↗
A new mutation in IVS-1 of the human beta globin gene causing beta thalassemia due to abnormal splicing.
A G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites. Only one of the two major cryptic sites was utilized in a cell-free splicing extract. The effects of this mutation on messenger RNA (mRNA) splicing are similar to that of another beta thalassemia gene with a G to C transition at the same position.
Base Sequence↗
Diagnosis of intermediate-duration and subacute organic mental disorders in abstinent alcoholics.
In a longitudinal study of 136 male alcoholics and controls, the authors found evidence of slowly reversible, subtle organic mental disorders in relation to prolonged abstinence from drinking by alcoholics. They suggest criteria for two new nosologic categories to describe these alcohol-related neuropsychiatric phenomena: (1) intermediate-duration organic mental disorder associated with alcoholism and (2) subacute organic mental disorder associated with alcohol abuse or alcoholism.
Adult↗
Prognostic significance of tumor grade in clinical trials of adjuvant therapy for breast cancer with axillary lymph node metastasis.
The prognostic significance of histologic tumor grade has been evaluated in 1537 women entered into the Ludwig Trials I-IV of adjuvant therapy for node-positive breast cancer. Tumor grade was determined on histologic review of primary tumor sections by two central review pathologists using a modification of the Bloom and Richardson grading system. The 5-year overall survival rates (+/- SE) were: Grade 1, 86% +/- 2; Grade 2, 70% +/- 2; and Grade 3, 57% +/- 2 (P less than 0.0001). This survival difference was seen in both premenopausal (P less than 0.0001) and postmenopausal (P less than 0.0001) women. Significant differences in disease-free survival (DFS) by tumor grade were also observed (P less than 0.0001). The tumor grade determined by the 75 contributing local clinic pathologists was also highly significant for predicting DFS and overall survival. Tumor grade remained a statistically significant prognostic factor for DFS (P less than 0.0001) and overall survival (P less than 0.0001) in multivariate analyses controlling for nodal status, tumor size, estrogen receptor status, menopausal status, age, peritumoral vessel invasion, and treatment assigned. In postmenopausal patients for whom adjuvant treatment was compared with no adjuvant therapy, the prognostic significance of tumor grade was modified by the effect of treatment. The presence of vessel invasion by primary tumor cells was a stronger predictor of early recurrence than was increasing tumor grade in postmenopausal patients who received no adjuvant therapy. The higher failure rates for patients with high-grade tumors was due to a larger number of failures in regional and visceral sites. Tumor grade can be determined by any pathologist and allows for selection of a subpopulation of breast cancer patients at high risk for early mortality.
Adult↗
A role for exon sequences and splice-site proximity in splice-site selection.
Analysis of the in vitro splicing products of RNA precursors containing tandem duplications of the 5' or 3' splice sites of human beta-globin IVS 1 revealed that exon sequences play an important role in the relative use of the duplicated sites. These studies also show that the proximity of the 5' and 3' splice sites is an important determinant in the selection of splice-sites. Deletion, substitution, or even subtle changes of exon sequences can alter the pattern of splice-site selection, and in many cases the splice site adjacent to the altered exon is not used. The relative use of the duplicated splice sites can also be altered by diluting the splicing extract, suggesting that factors involved in splice-site selection are limiting.
Base Sequence↗
Handling the media. Your patient on the Evening News.
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Allied Health Personnel↗
Intron sequences involved in lariat formation during pre-mRNA splicing.
We have shown that lariat formation during in vitro splicing of several RNA precursors, from Drosophila to man, occurs at a unique and identifiable but weakly conserved site, 18 to 37 nucleotides proximal to the 3' splice site. Lariat formation within an artificial intron lacking a normal branch-point sequence occurs at a cryptic site a conserved distance (approximately 23 nucleotides) from the 3' splice site. Analysis of beta-thalassemia splicing mutations revealed that lariat formation in the first intron of the human beta-globin gene occurs at the same site in normal and mutant precursors, even though alternate 5' and 3' splice sites are utilized in the mutants. Remarkably, cleavage at the 5' splice site and lariat formation do not occur when the precursor contains a beta-thalassemia deletion removing the polypyrimidine stretch and AG dinucleotide at the 3' splice site. In contrast, a single base substitution in the AG dinucleotide blocks cleavage at the 3' splice site but not at the 5' site.
Animals↗