Use of sevoflurane inhalation sedation for outpatient third molar surgery.

This study attempted to determine if sevoflurane in oxygen inhaled via a nasal hood as a sole sedative agent would provide an appropriate level of deep sedation for outpatient third molar surgery. Twenty-four patients scheduled for third molar removal were randomly assigned to receive either nasal hood inhalation sevoflurane or an intravenous deep sedation using midazolam and fentanyl followed by a propofol infusion. In addition to measuring patient, surgeon, and dentist anesthesiologist subjective satisfaction with the technique, physiological parameters, amnesia, and psychomotor recovery were also assessed. No statistically significant difference was found between the sevoflurane and midazolam-fentanyl-propofol sedative groups in physiological parameters, degree of amnesia, reported quality of sedation, or patient willingness to again undergo a similar deep sedation. A trend toward earlier recovery in the sevoflurane group was identified. Sevoflurane can be successfully employed as a deep sedative rather than a general anesthetic for extraction of third molars in healthy subjects.

A developmentally regulated kinesin-related motor protein from Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.

The use of leisure activities in a therapeutic community.

This paper is concerned with the use of social and leisure activities as part of nursing work in a therapeutic community. It describes this element in the therapeutic programme of the Cassel Hospital. There is a brief introduction to the general principles of the therapeutic community and then to the nurses' role at the Cassel Hospital. The work in the sphere of social and leisure activities is seen as consistent with these principles. The structures enabling social and leisure activities to be integrated into the work of the hospital are described. Therapeutic opportunities are outlined, which arise when nurses work alongside patients in the course of such activities. This also serves to illustrate therapeutic community practice. There follows a discussion of the clinical significance of the voluntary element in this area of practice and of the related problems. Jacques' conceptualization of work and resistance to work are used at a number of points to link issues raised in the paper to existing psychodynamic theory.

The role of the carbohydrate chains of Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase for enzyme activity.

Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2,6 sialyltransferase, EC 2.4.99.1) is a glycoprotein containing carbohydrate chains of the complex type (Jamieson, J.C. (1989) Life Sci. 43, 691-697). The carbohydrate chains may be important for controlling the expression of sialyltransferase catalytic activity during transit of the enzyme from the rough endoplasmic reticulum to the Golgi complex where it is active as a membrane bound enzyme anchored to the luminal face. To study the role of the carbohydrate chains of sialyltransferase for enzyme activity, conditions were established in which the native enzyme was deglycosylated with N-Glycanase and endo F. It was found that Glycanase removed the carbohydrate chains from native sialyltransferase, but methanol or ethanol had to be present for rapid and complete deglycosylation. Presence of methanol or ethanol were not essential for removal of carbohydrate chains with endo F. There was a correlation between the loss of catalytic activity of sialyltransferase with increased deglycosylation. After deglycosylation with Glycanase for 18 h catalytic activity was largely eliminated and there was a reduction in molecular mass of about 5 kDa compared to the untreated enzyme when examined by immunoblot analysis; this reduction was identical to that found when the denatured enzyme was deglycosylated with Glycanase. At shorter times of incubation partially deglycosylated forms of the enzyme were detected. Complete deglycosylation of native or denatured sialyltransferase with endo F could not be achieved. However, incubation with endo F for 24 h resulted in a loss of catalytic activity of about 60%. Immunoblot analysis showed the presence of three forms of the enzyme corresponding in molecular mass to the native and deglycosylated enzyme and a third form corresponding to a partially deglycosylated enzyme. Sialyltransferase was also subjected to sequential treatment with exoglycosidases. Removal of NeuAc and Gal had little effect on catalytic activity, but subsequent removal of GlcNAc resulted in a significant loss in catalytic activity suggesting that the presence of the trimannose core with GlcNAc attached is important for the expression of catalytic activity. The presence of organic solvents during deglycosylation with Glycanase may be a useful method that can be applied to other glycoproteins.

Sialyltransferase: a novel acute-phase reactant.

1. Proteins that are released into the circulation in elevated amounts in injured mammals are referred to as acute-phase reactants. Most are liver synthesized glycoproteins of the secretable type. However, Gal-beta(1-->4)-GlcNAc-alpha(2-->6)-sialyltransferase (EC 2.4.99.1) is a novel acute-phase reactant since it is a Golgi membrane-bound enzyme rather than a secretable glycoprotein. 2. The role of glucocorticoids and cytokines in the control of synthesis and expression of acute-phase glycoproteins, including sialyltransferase, is discussed. 3. The acute-phase behaviour of Gal-beta(1-->4)-GlcNAc-alpha(2-->6)-sialyltransferase is dependent on the release of the enzyme from the Golgi in the acute-phase state. The mechanism of release of a catalytically active form of the enzyme is described.

Evidence for the role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from rat and mouse liver in whole-cell systems.

1. Sialyltransferase is a liver Golgi membrane-bound enzyme that is released from the liver under conditions of experimental inflammation. Previous work showed that the action of a cathepsin D-like proteinase was responsible for release of the enzyme from isolated Golgi membranes. This study shows that the same enzyme is responsible for release of sialyltransferase in whole-cell systems. 2. Gal beta 1-4GlcNAc alpha 2-6sialyltransferase (EC 2.4.99.1) was secreted from slices of rat and mouse liver into the incubation medium with larger amounts of activity being secreted from slices of liver from animals suffering from experimental inflammation. 3. The presence in the incubation medium of the cathepsin D proteinase inhibitor, pepstatin A, at 10(-4) M was sufficient to inhibit the release of sialyltransferase into the medium by about 60% after a 6 hr incubation. 4. The release of albumin and alpha 1 acid glycoprotein from rat liver slices, was not affected by the presence of pepstatin A, indicating that the proteinase inhibitor did not affect the synthesis and secretion of typical secretable proteins by the liver. 5. Intraperitoneal injections of pepstatin A into mice prior to preparation of liver slices also resulted in a significant reduction of the secretion of sialyltransferase into the incubation medium. 6. The results from these studies support the idea that a cathepsin D-like proteinase is responsible for the release of sialyltransferase into the extracellular space in whole cells in the rat and the mouse.

Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells.

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.

Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae.

The FUS1 gene of Saccharomyces cerevisiae is transcribed in a and alpha cells, not in a/alpha diploids, and its transcription increases dramatically when haploid cells are exposed to the appropriate mating pheromone. In addition, FUS1 transcription is absolutely dependent on STE4, STE5, STE7, STE11, and STE12, genes thought to encode components of the pheromone response pathway. We now have determined that the pheromone response element (PRE), which occurs in four copies within the FUS1 upstream region, functions as the FUS1 upstream activation sequence (UAS) and is responsible for all known aspects of FUS1 regulation. In particular, deletion of 55 bp that includes the PREs abolished all transcription, and a 139-bp fragment that includes the PREs conferred FUS1-like expression to a CYC1-lacZ reporter gene. Moreover, three or four copies of a synthetic PRE closely mimicked the activity conferred by the 139-bp fragment, and even a single copy of PRE conferred a trace of activity that was haploid specific and pheromone inducible. In the FUS1 promoter context, four copies of the synthetic PRE inserted at the site of the 55-bp deletion restored full FUS1 transcription. Sequences upstream and downstream from the PRE cluster were important for maximal PRE-directed expression but, by themselves, did not have UAS activity. Other yeast genes with PREs, e.g., STE2 and BAR1, are more modestly inducible and have additional UAS elements contributing to the overall activity. In the FUS1 promoter, the PREs apparently act alone to confer activity that is highly stimulated by pheromone.

The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames.

Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.

Identification of a kinesin-like microtubule-based motor protein in Dictyostelium discoideum.

Dictyostelium discoideum, a unicellular eukaryote amenable to both biochemical and genetic dissection, provides an attractive system for studying microtubule-based transport. In this work, we have identified microtubule-based motor activities in Dictyostelium cell extracts and have partially purified a protein that induces microtubule translocation along glass surfaces. This protein, which sediments at approximately 9S in sucrose density gradients and is composed of a 105 kd polypeptide, generates anterograde movement along microtubules that is insensitive to 5 mM NEM (N-ethyl-maleimide) but sensitive to 200 microM vanadate, and has similar nucleotide-dependent microtubule binding properties to those of kinesins purified from mammals, sea urchin and Drosophila. This kinesin-like molecule from Dictyostelium, however, is immunologically distinct from bovine and squid neuronal kinesins and supports microtubule movement on glass at four-fold greater velocities (2.0 versus 0.5 microns/sec). Furthermore, AMP-PNP (adenylyl imidodiphosphate), which promotes attachment of previously characterized kinesins to microtubules, decreases the affinity of the Dictyostelium kinesin homolog for microtubules. Thus, an AMP-PNP-induced rigor binding may not be a characteristic of kinesins from lower eukaryotes.

Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae.

We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells. Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1. In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone. To investigate the function of the Fus1 protein, we created fus1 null mutants. In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein. Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor.

Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the alpha mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by alpha cells and is transcribed only in alpha cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First, wild-type Ste3 product is required for response to the pheromone: mutants with any one of five different ste3 mutations are unresponsive to a factor. Second, a hybrid Ste3-beta-galactosidase protein encoded by a STE3-lacZ gene fusion fractionates to the particulate fraction of yeast cell extracts, suggesting that Ste3 is a membrane protein. Finally, the DNA sequence of STE3, which we report here, encodes a protein of 470 amino acid residues that contains seven distinct hydrophobic segments of sufficient length to span a lipid bilayer.

Programmed aging or error catastrophe? An examination by two-dimensional polyacrylamide gel electrophoresis.

We have examined newly synthesized proteins in the young adult and in older populations of the nematode Caenorhabditis elegans using two-dimensional polyacrylamide gel electrophoresis (2D PAGE). A temperature-sensitive mutant strain, DH26, with a mean life span of about 15 days, under our conditions, was used to block progeny development. Nematodes of several different ages were pulse-labeled for 5 h, in vivo, with 35S-labeled E. coli, A subsequent 30-min chase with unlabeled E. coli served to rid the worms of endogenous labeled E. coli proteins. We resolve 700 or more proteins by 2D PAGE polyacrylamide gel electrophoresis of extracts of young nematodes. The patterns of these proteins are highly reproducible in comparisons of independent repeats of identical experiments. No new major proteins are synthesized at any time during the adult phase (4-22 days) nor are any of the most abundant proteins not made during this period. At our level of detectability (estimated as a satellite spot containing 4% of the amount of label in a major spot) we see no misincorporation of radioactive amino acids into newly synthesized proteins. These data are inconsistent with predictions by any one of several, so called, "error catastrophe" models of senescence and also show that modulation of the highest abundancy classes of proteins are also not involved in senescence.