An investigation of the effect of 2 sedation regimens on patient mood state following upper extremity surgery using local anesthesia.

Sedation techniques for patients undergoing minor outpatient surgery frequently include a variety of intravenous agents. The present study was designed to look for differential effects of 2 different sedation regimens on perioperative mood states. Twenty-two patients undergoing upper extremity surgery using local anesthesia were randomized to receive either propofol or midazolam intravenously for intraoperative sedation. Subjects were asked to complete a Profile of Mood States survey before and after surgery. The results of this survey were examined for differences in mood between the 2 groups that may be attributable to differences in drug effect. No significant differences were identified between propofol or midazolam regarding their effect on patient mood. Patients in both groups experienced a reduction in perioperative anxiety.

The accumulation of cadmium by the yellow pond lily, Nuphar variegatum, in Ontario peatlands.

Cadmium concentrations of a common macrophyte, the yellowpond lily (Nuphar variegatum) were investigated from peatlands with arange in pH (4.4-6.3), alkalinity (0-181 microeq/L Ca), DOC (5.1-16.8 mg/L), and sediment organic content (20-88%). Cd concentrations inNuphar ranged from 0.3 to 1.51 microg/g in the leaves and from 0.46 to1.51 microg/g in the petioles, and was significantly higher in the petiolesthan in the leaves (p = 0.014; t-Test). Significant and negativecorrelations between Nuphar leaf Cd and pH (r2 =0.76; p < 0. 001), alkalinity (r2 = 0.41; p =0.034), and DOC (r2 = 0.46; p = 0. 022) wereobtained. In addition, a significant and negative correlation was foundbetween Nuphar petiole Cd and pH (r2 = 0.46; p= 0.023). These results indicate that the leaves of Nupharfound in peatlands of low pH, low alkalinity, and low DOC, and the petiolesof Nuphar found in peatlands of low pH are more susceptible toaccumulating potentially toxic levels of Cd. The organic content of thepeatland sediments was not significantly correlated with either the leaf orpetiole Cd concentration. Nuphar is an important food source for manywetland animals; any Cd that is present in these plants may be passed ontoother trophic levels because diet is considered to be the major source of Cdto animals.

Molecular mapping of the Cromer blood group Cra and Tca epitopes of decay accelerating factor: toward the use of recombinant antigens in immunohematology.

Cromer blood group antigens reside on the complement regulatory protein decay accelerating factor (DAF, CD55). This glycosyl-phosphatidylinositol-anchored glycoprotein is widely distributed, especially among cell types in contact with plasma. Numerous Cromer blood group antigens have been defined using alloantibodies induced by transfusion or pregnancy. However, few pairs of antithetical antigens have been described in this system, presumably because of the rarity of the low-frequency alleles. Analysis of polymerase chain reaction-amplified genomic DNA showed that the Cr(a-) phenotype has a Ala193-->Pro substitution in short consensus repeat 4 (SCR4) of DAF, and the Tc(a-b+) phenotype has a Arg18-->Leu substitution in SCR1 of DAF. The locations of Cra and Tca epitopes were confirmed by analysis of Chinese hamster ovary cell transfectants expressing a Cr(a-) allele-specific transfectant and a chimeric protein containing only SCR1 of DAF, respectively. Overall, these studies further show the usefulness of an approach based on recombinant proteins in mapping blood group antigen epitopes and identifying blood group antibodies.

Molecular basis of reduced or absent expression of decay-accelerating factor in Cromer blood group phenotypes.

The human erythrocyte blood group system Cromer consists of high-incidence and low-incidence antigens that reside on decay-accelerating factor (DAF; CD55), a glycosyl-phosphatidylinositol-anchored membrane protein that regulates complement activation on cell surfaces. In the Cromer phenotypes Dr(a-) and Inab there is reduced or absent expression of DAF, respectively. This study investigated the molecular basis of the reduced DAF expression by polymerase chain reaction amplification of genomic DNA and RNA/cDNA obtained from Epstein-Barr virus-transformed lymphoblastoid cell lines. Sequence analysis of the Inab propositus showed a single nucleotide substitution in exon 2 of the DAF gene and at the corresponding position in the cDNA, G314-->A resulting in Trp53-->Stop. This truncation near the amino terminus explains the complete absence of surface DAF in the Inab phenotype. A similar analysis was performed for two Dr(a-) individuals, including KZ, who was previously reported to be Inab phenotype but is now shown by immunochemical and serologic methods to be Dr(a-) phenotype. A single nucleotide change was found in exon 5 of the DAF gene, C649-->T resulting in Ser165-->Leu, which we had previously shown to lead to loss of the Dra epitope. However, two species of cDNA were found, one encoding full-length DAF with the single amino acid change and the more abundant species having a 44-nucleotide deletion. The 44 nucleotide deletion includes the single polymorphic site, which creates a cryptic branch point in the Dr(a-) allele that leads to use of a downstream cryptic acceptor splice site. This shifts the reading frame and leads to a premature stop codon that precludes membrane anchoring. Thus, the single point mutation in the Dr(a-) phenotype results in a novel use of alternative splicing and provides a molecular explanation for both the antigenicity and the reduced DAF expression seen in this phenotype.

Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhD-negative individuals.

The Rh blood group system plays a major role in immune and nonimmune hemolytic states. Although an Rh cDNA has been previously cloned, there is no information on which Rh antigenic protein it encodes. Using polymerase chain reaction (PCR) amplification, we have identified this original Rh clone, here designated Rh21, and an additional Rh cDNA clone, Rh13, that is 96% nucleotide- and 92% amino acid-identical to Rh21, with the substitutions scattered throughout the sequence. A molecular genetic approach was used to match this Rh clone with an Rh specificity. The mRNA transcript for Rh13 was present in reticulocytes from RhD-positive individuals, but was absent from the reticulocytes of RhD-negative individuals. Using conventional screening of genomic libraries, as well as PCR cloning, partial genomic clones for these two Rh cDNAs were obtained. Based on PCR analysis and Southern blots, the Rh21 gene was present in all individuals, but an intact Rh13 gene was only present in RhD-positive and not RhD-negative individuals. Thus, by correlating the presence of Rh mRNA and gene sequences with individual Rh phenotypes, we were able to establish that the new Rh13 cDNA clone represents the RhD protein.

Dr(a-) polymorphism of decay accelerating factor. Biochemical, functional, and molecular characterization and production of allele-specific transfectants.

The Dra antigen belongs to the Cromer-related blood group system, a series of antigens on decay accelerating factor (DAF), a glycosyl-phosphatidylinositol-anchored membrane protein that protects host cells from complement-mediated damage. We studied the rare inherited Dr(a-) phenotype to ascertain the associated biochemical and functional changes in DAF and to characterize the basis for this polymorphism. Radioimmunoassay assay and flow cytometric analysis of Dr(a-) erythrocytes demonstrated 40% of normal surface expression of DAF but normal levels of several other glycosyl-phosphatidylinositol-anchored proteins, distinguishing this phenotype from that of paroxysmal nocturnal hemoglobinuria. Western blots confirmed this reduced DAF expression and indicated a slightly faster mobility of the molecule on SDS-PAGE. Despite the reduced DAF expression, Dr(a-) erythrocytes functioned normally in the complement lysis sensitivity assay. Utilization of the polymerase chain reaction to amplify mononuclear cell genomic DNA from three unrelated Dr(a-) individuals demonstrated that a point mutation underlies the Dr(a-) phenotype: a C to T change in nucleotide 649 resulting in a serine165 to leucine change. This defines the Drb allele of DAF, which can be distinguished from Dra by a Taq I restriction fragment length polymorphism. We created transfected Chinese hamster ovary cell lines expressing either the Dra or the Drb allelic form of DAF. These allele-specific transfectants were tested by inhibition of hemagglutination or flow cytometry and confirmed the specificity of anti-Dra alloantisera. The allele-specific transfectants could form the basis of a new serological approach to immunohematology.

Structure of the gene for human complement protein decay accelerating factor.

Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane protein that is part of the regulators of complement activation (RCA) gene family located on human chromosome 1, band q32. These proteins, beginning at their amino terminus, consist largely of multiple copies of an approximately 60 amino acid short consensus repeat (SCR). A DAF cDNA clone was used to identify overlapping bacteriophage genomic clones. The human DAF gene spans approximately 40 kb and consists of 11 exons. The length of these exons and introns varies considerably, with the exons ranging from 21 to 956 bp and the introns ranging from approximately 0.5 to 19.8 kb. SCR I, II, and IV are all encoded by single exons; however, SCR III is encoded by two separate exons, with the splice junction occurring after the second nucleotide of the codon for the glycine residue at position 34 of the consensus sequence. This feature has also been found in CR1, CR2, membrane cofactor protein, and murine factor H. Following the SCR in DAF is a 76 amino acid serine/threonine-rich domain encoded on three separate exons. Exon 10 encodes the Alu family sequence that has been found as an insert in a minor class of DAF cDNA, thus indicating that this mRNA arises by standard alternative splicing. The last DAF exon, which comes after the largest intron of 19.8 kb, encodes the hydrophobic carboxy terminus and the 3'UT region. The nature of the signal that directs posttranslational attachment of a glycophospholipid anchor to DAF is not known, but that signal is apparently spread over three exons and greater than 20 kb. An analysis of the DAF gene provides additional evidence for the common evolutionary heritage of the RCA gene family. The exon/intron structure of this gene will facilitate experiments aimed at understanding the functions of the various domains of DAF.