Neuroleptic malignant syndrome in pregnancy.

BACKGROUND: Neuroleptic malignant syndrome can be a serious neurologic complication of drug therapy during pregnancy. CASE: A young woman was admitted to the intensive care unit with worsening varicella pneumonia. After being given haldol for agitation, she developed fever, increasing agitation, rigidity, tachycardia, and tremors; she was diagnosed as having neuroleptic malignant syndrome. She was treated successfully with bromocriptine and dantrolene. CONCLUSION: Despite the common use of antipsychotic medications, neuroleptic malignant syndrome is seen infrequently during pregnancy. The diagnosis can be difficult to make, but if suspected, it can be treated successfully.

A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport.

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the antidiabetic effects of the glitazone drugs in humans are due to activation of the alpha (NR1C1) and gamma (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the delta (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPARdelta agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPARdelta agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

Cost consequences of elimination of the routine group B streptococcus culture at a teaching hospital.

OBJECTIVE: To evaluate the cost consequence of the elimination of routine Group B streptococcus (GBS) cultures in pregnancy utilizing risk factor assessment management recommendations of the Center for Disease Control. METHODS: This retrospective study cohort population included all delivering patients from June 1, 1996, to May 31, 1997, managed by the Morbidity Mortality Weekly Report (MMWR) guidelines May 31, 1996, for GBS in pregnancy compared to the previous 29 months cohort from January 1, 1994, to May 31, 1996, managed with routine GBS cultures done at 35-37 weeks. RESULTS: Of the 7,681 culture management control cohort patients, there were four neonates with culture-positive GBS sepsis (1/1,900). The cost for detection of a single positive culture in an affected neonate was $8,627 ($34,509/4) and there were 2,875 personnel hours expended. In contrast, of the 2,011 patients in the risk factor management cohort, there were two cases of neonatal GBS sepsis ($111,005). The cost for detection of a positive culture in an affected neonate was $1,579 ($3,159/2) and there were 263 personnel hours expended in the risk factor management group. In spite of these significant laboratory savings, we noted a concurrent increase in the total cost in the newborn nursery for septic work-ups and treatment from $2.4 million to $3.1 million. CONCLUSION: Risk assessment management of GBS provided a savings of both money ($7,048/positive neonatal culture) and laboratory time (586 personnel hours/positive neonatal culture). However, these savings were more than offset by cost increases occurring in the newborn nursery ($400,000), demonstrating the necessity of practice patterns to undergo concurrent evaluation to verify cost savings and prevent shifting of expenses.

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression.

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.

Recombinant proteins for genetic disease.

The era of molecular biology has led to the development of powerful tools capable of generating therapeutics for genetic disorders. Although there is much current emphasis placed on the development of 'gene therapy' for human disease, developments in the production and availability of recombinant proteins are likely to have a more substantial impact on genetic disease in the short term. The clinical evaluation of recombinant or purified proteins serves as an initial 'proof of principle' of gene-based therapies and thus should expedite advances in this area. Current examples include the use of bovine adenosine deaminase for a form of severe combined immune deficiency (SCID) (Hilman BC, Sorensen RU. Management options: SCIDS with adenosine deaminase deficiency. Ann Allergy 72: 1994: 395-403) and glucocerebrosidase for Gaucher disease (Niederau C, vom Dahl S, Haussinger D. First long-term results of imiglucerase therapy of type 1 Gaucher disease. Eur J Med Res 1998: 3: 25-30). The success of these two enzyme replacement regimes in human clinical trials has been a main impetus for the development of gene-based therapies for these disorders. The 'molecularization of medicine' has led to a more thorough understanding of the molecular basis of disease and disease pathogenesis. The availability of recombinant proteins and the development of appropriate delivery systems will result in more widespread use of these agents. Protein engineering will generate agents with novel functions as is already witnessed with the generation of fusion proteins. This review will highlight advances in the use of recombinant proteins for genetic disease and future potential uses of recombinant proteins.

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.

Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control.

The PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that participates in the second step of the splicing reaction. It was found recently that the yeast PRP17 gene is identical to the cell division cycle CDC40 gene. The PRP17/CDC40 gene codes for a protein with several copies of the WD repeat, a motif found in a large family of proteins that play important roles in signal transduction, cell cycle progression, splicing, transcription, and development. In this report, we describe the identification of human, nematode, and fission yeast homologues of the PRP17/CDC40 gene of S. cerevisiae. The newly identified proteins share homology with the budding yeast protein throughout their entire sequence, with the similarity being greatest in the C-terminal two thirds that includes the conserved WD repeats. We show that a yeast-human chimera, carrying the C-terminal two thirds of the hPRP17 protein, is able to complement the cell cycle and splicing defects of a yeast prp17 mutant. Moreover, the yeast and yeast-human chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, providing evidence that these proteins are spliceosome-associated. These results show the functional conservation of the Prp17 proteins in evolution and suggest that the second step of splicing takes place by a similar mechanism throughout eukaryotes.

The process of change in couples therapy: a qualitative investigation.

Open-ended interviews with 24 couples therapy clients regarding their experience of the process of change revealed shifts in clusters of affect, communication, and cognition. Six additional contextual preconditions for change were also identified. The change process within couples was uniformly reported to be gradual.

Examining the multifaceted notion of isomorphism in marriage and family therapy supervision: a quest for conceptual clarity.

The notion of isomorphism has been recommended as a conceptual framework to guide the practice of marriage and family therapy (MFT) supervision. The term is frequently cited in the MFT training literature but is often used in different ways. A panel of MFT supervisors rated the importance and relevance to both therapy and supervision of a large pool of variables. The majority of variables were found to be equally relevant or isomorphic to the domains of MFT and MFT supervision. A qualitative interview with a small subset of the panelists suggested that the concept, to varying degrees, has influenced their work as supervisors. The implications of the results for theory development, research and supervisory practice are discussed.

Murine mucopolysaccharidosis type I: targeted disruption of the murine alpha-L-iduronidase gene.

Mucopolysaccharidosis type I (MPS I) is considered to represent the prototypical mucopolysaccharide storage disorder. Although a spectrum of severity is seen within the MPS I subgroup, Hurler syndrome represents the most severe and frequent manifestation of MPS I. We describe here the generation of a murine model for Hurler syndrome by targeted disruption of the murine Idua gene. Homozygous Idua -/- mice have no detectable alpha-L-iduronidase enzyme activity and show increased urinary glycosaminoglycan levels. Although normal appearing at birth, Idua -/- mice develop a flattened facial profile and thickening of the digits discernible by 3 weeks of age. No obvious growth deficiency nor mortality is seen within the first 20 weeks of life. Radiographs reveal anterior flaring of the ribs and thickening of the facial bones as early as 4 weeks of age with more extensive dysostosis detectable by 15 weeks of age. At 4 weeks of age, lysosomal storage is noted primarily within reticuloendothelial cells with abundant lysosomes noted in Kupffer cells, splenic sinusoidal lining cells, and glial cells. More widespread lysosomal storage is noted by 8 weeks of age in hepatocytes, chondrocytes, neurons, as well as renal tubular cells. Thus, targeted disruption of the murine Idua locus has produced a murine strain representative of the severe form of MPS I. This model should permit detailed evaluation of the pathophysiology of lysosomal storage disorders and provide a small animal model for the testing and development of enzyme replacement and gene therapy regimes.

Expression of glutamyl-tRNA reductase in Escherichia coli.

The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA. Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step. In Escherichia coli, GTR is the gene product of hemA. BL21(DE3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37 degrees C), presence of betaine and sorbitol (no change) and time of induction (2h). GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels. Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNAglu. Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme. The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx. 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA. This expression was higher in the presence of tRNAglu. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. We conclude that GTR is present in both high molecular weight species. Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117-126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA. These possibilities are being investigated.

Characterization of a hemA/hemE mutant of E. coli and regulation of hemE.

Uroporphyrinogen III is the committed intermediate common to heme and siroheme biosynthesis in E. coli. Uroporphyrinogen III decarboxylase is the first enzyme at the branch point which commits to heme synthesis. A hemin-permeable hemA mutant which could grow on 5-aminolevulinic acid (ALA) or hemin, was mutagenized to give a double mutant, 10L2-1. The second mutation which was identified as hemE because it was mapped to 90.1 min. by F' and Hfr mapping and P1 transduction, accumulated uroporphyrin and had no uroporphyrinogen decarboxylase activity. This mutation could be complemented with a plasmid harboring the hemE gene of Synechococcus. The complemented strain could grow on ALA and accumulated coproporphyrin and protoporphyrin but not uroporphyrin. The E. coli hemE gene was cloned by transducing 10L2-1 with an E. coli genomic library in lambda gt11. hemE with upstream regions of various sizes was cloned in front of a promoterless CAT gene. Good growth on chloramphenicol (25-75 micrograms/ml) depended on a promoter within 152 bp upstream of the hemE structural gene start of translation site. In addition, this construct could complement the hemE requirement of 10L2-1 as well as allow it to grow on chloramphenicol. Addition of hemin did not inhibit this growth and therefore it appears that it does not affect the hemE promoter. The hemE structural gene alone allowed good growth on 10 micrograms/ml but poor growth on 25 micrograms/ml chloramphenicol, suggesting that there is a weak promoter within hemE for a downstream ORF. Quantitation of CAT protein in these strains showed a weak promoter within hemE, a promoter 152 bp upstream of hemE and another promoter within 1.3 kb upstream of hemE. The 1.3 kb region contains an ORF 40 bp upstream of hemE, thus suggesting that hemE is part of an operon.

Heating unsaturated fatty acids in air produces hemagglutinins.

When mono-unsaturated fatty acids are heated in air, they form hemagglutinins. When the double bond is delta-6,7 or delta-9,10, the titer is higher than for delta-11,12. Stearic acid does not become a hemagglutinin on heating. Hydroxy-monounsaturated fatty acids, ricinoleic (cis-12-OH-delta-9) and ricinelaidic (trans-12-OH-delta-9) are not hemagglutinins unless they are heated. Oleic acid (delta-9-octadecenoic acid, OA) has a very low agglutination titer but lyses red cells at higher concentrations. Rabbit and rat erythrocytes (RBC) give the highest titers but RBCs of other species are also agglutinated. OA was chosen for further study. Its specific titer against rat RBCs increases with time of heating in air. Thin-layer chromatography (TLC) and mass spectroscopy (MS) show that higher molecular weight compounds are formed and that activity is associated with these materials. Synthetic (oxidation of oleic acid with tert-butyl peroxide) and commercial preparations of oleic acid dimers (Emery and Unichema) and a commercial preparation of oleic trimer mixed with polymer (Emery) have high hemagglutination titers against rat erythrocytes. A cyclic, long-chain dicarboxylic acid, 5(6)-carboxy-4-hexyl-2-cyclohexene-1-octanoic acid (Westvaco) gives a very low titer unless heated and no lysis. Sialidase treatment of the red cells increases the titer. Removal of cations does not alter the titer but addition of Ca2+ or Mg2+ lowers the titer. Light microscopy was used to characterize and visualize the agglutination process with rat RBCs. Agglutination without lysis or fusion is observed for low concentrations of heated oleic acid and C-18 dimers and trimer-polymer preparations, and no large vesicles are seen. We conclude that the oligomeric fatty acids with two or more hydrophobic chains of seven or more carbons are agglutinins at physiological pH. Agglutination by dimer may be the result of the its two hydrophobic side chains inserting into adjacent RBC membranes or the result of dimer inserting completely into RBC membranes and altering their properties. The carboxyl groups may also play a role in the process by interacting with polar headgroups in the RBC membrane.

5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA.

hemA and hemM, which are 213 bp apart and divergently transcribed, were separately cloned. We found that hemA is required for 5-aminolevulinic acid (ALA) synthesis in two ALA- auxotrophs. Overexpression of hemM alone did not produce ALA. More ALA was produced by strains harboring a plasmid with both hemA and hemM than by those with hemA alone. We conclude that hemA alone is required for ALA synthesis but hemA and hemM are required for maximal ALA synthesis.

The therapist who is perceived as "spiritually correct": strategies for avoiding collusion with the "spiritually one-up" spouse.

Marital and family therapists who are perceived by the community as having a strong spiritual orientation face unique and difficult challenges. Clients who seek their help often bring a mix of expectations that can create a situation in which the therapist may struggle with multiple roles, some not of his or her choosing. These expectations are especially challenging when one spouse views him-or herself as "spiritually one-up" and wants the therapist to form a spiritually based coalition against the other partner. This paper describes the various dilemmas that therapists may face when clients perceive them as having spiritual expertise and presents strategies that can be used for avoiding these dilemmas. A case study illustrates the use of these approaches.

Antithrombotic actions of aspirin in the horse.

The antithrombotic effects of aspirin at two dose rates (4 mg/kg and 11 mg/kg bodyweight [bwt] were evaluated in normal, healthy ponies by measuring template bleeding time. Inhibition of platelet aggregation in response to adenosine diphosphate (ADP) and collagen was evaluated and cyclo-oxygenase activity was monitored by radioimmunoassay of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 (TXA2). TXB2 was measured in serum and platelet rich plasma. Bleeding time was prolonged significantly until 48 h after treatment at 12 mg/kg bwt and until 4 h at the lower dose rate. Synthesis of TXB2 and collagen induced aggregation were diminished for much greater periods with similar results at each of the dose rates. The prolonged effects of aspirin on platelet function occurred in spite of a very short plasma half-life of aspirin, because of its irreversible action on platelet cyclo-oxygenase. The results show that low dose aspirin has a potential role in antithrombotic therapy in horses although the relationship between skin bleeding time in normal horses and improvement of clinical conditions requires further research and evaluation in clinical trials. TXB2 measurement appears to overestimate the duration of antithrombotic effects of aspirin in vivo.

Cloning and structure of the hem A gene of Escherichia coli K-12.

An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24h. The cloned sequence appears to encode a 46-kDa protein, which when produced in the maxicell procedure is processed to a 41-kDa protein as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The amino acid sequence of the cloned gene product shows no significant homologies with any cloned ALA synthase, nor with any protein, in two E. coli databanks. A second cloned gene fragment, which has its coding region 34 bp away from the coding region of the gene that complements hemA, has been identified as part of protein release factor 1(RF1), thus confirming the location of hemA at min 26.7 and mapping it precisely near RF1. We have shown that E. coli utilizes the intact five-carbon chain of glutamate for the synthesis of ALA [Li et al., J Bacteriol. 171 (1989b) 2547-2552].