Automated tracheal intubation in an airway manikin using a robotic endoscope: a proof of concept study.

Robotic endoscope-automated via laryngeal imaging for tracheal intubation (REALITI) has been developed to enable automated tracheal intubation. This proof-of-concept study using a convenience sample of participants, comprised of trained anaesthetists and lay participants with no medical training, assessed the performance of a robotic device for the insertion of a tracheal tube into a manikin. A prototype robotic endoscope device was inserted into the trachea of an airway manikin by seven anaesthetists and seven participants with no medical training. Each individual performed six device insertions into the trachea in manual mode and six in automated mode. The anaesthetists succeeded with 40/42 (95%) manual insertions (median (IQR [range]) 17 (12-26 [4-132]) s) and 40/42 (95%) automated insertions (15 (13-18 [7-25]) s). The non-trained participants succeeded in 41/42 (98%) manual insertions (median (IQR [range]) 18 (13-21 [8-133]) s) and 42/42 (100%) automated insertions (16 (13-23 [10-58])] s). The duration of insertion did not differ between groups. An effect of increasing experience was observed in both groups in manual mode. A Likert scale for 'ease of use' (0 = very difficult to 10 = very easy) showed similar results within the two groups; the mean (SD) was 5.9 (2.1) for the anaesthetists and 6.9 (1.3) for the non-trained participants. We have successfully performed the first automated tracheal device insertion in a manikin with comparable results in a convenience sample of anaesthetists and lay participants with no medical training.

Prevalence and risk factors for Staphylococcus aureus colonization in individuals entering maximum-security prisons.

To assess the prevalence and risk factors for colonization with Staphylococcus aureus in inmates entering two maximum-security prisons in New York State, USA, inmates (N=830) were interviewed and anterior nares and oropharyngeal samples collected. Isolates were characterized using spa typing. Overall, 50·5% of women and 58·3% of men were colonized with S. aureus and 10·6% of women and 5·9% of men were colonized with MRSA at either or both body sites. Of MSSA isolates, the major subtypes were spa type 008 and 002. Overall, risk factors for S. aureus colonization varied by gender and were only found in women and included younger age, fair/poor self-reported general health, and longer length of prior incarceration. Prevalence of MRSA colonization was 8·2%, nearly 10 times greater than in the general population. Control of epidemic S. aureus in prisons should consider the constant introduction of strains by new inmates.

Isolation of a nitric oxide inhibitor from mammary tumor cells and its characterization as phosphatidyl serine.

Macrophages from mice bearing large D1-DMBA-3 mammary tumors have a decreased capacity to kill tumor targets. This effect is due to an impaired ability to produce nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. Here we report that the DA-3 tumor cell line, derived from the in vivo adenocarcinoma D1-DMBA-3, produces a factor that inhibits both NO production/release and cytotoxicity of LPS-activated peritoneal exudate macrophages (PEM). However, other complex macrophage functions such as phagocytosis, superoxide production, mitochondrial dehydrogenase activity, and synthesis of proteins were not reduced by this factor. The NO inhibitor has been found to be lipid in nature. Lipid extracts from DA-3 cell culture supernatants were purified by repeated silica gel column chromatography. The active molecule was unambiguously characterized as phosphatidyl serine (PS) by fast atom bombardment tandem mass spectrometry. Preliminary results indicate a lack of induced NO synthase (iNOS) activity in the lysates of LPS-activated PEM pretreated with PS. The ubiquity of PS in the inner leaflet of biological membranes and its NO inhibitory property, suggest that this phospholipid may be one of the long elusive molecules responsible for regulating physiological levels of NO in the host and hence preventing cellular dysfunction and/or tissue damage. Furthermore, the possible overexpression and shedding of PS by DA-3 tumor cells may represent a novel mechanism to impair macrophage cytotoxicity, a host function that contributes to the protection against developing neoplasms.

Coordinate regulation of beta-lactamase induction and peptidoglycan composition by the amp operon.

The amp operon, which is located on the Escherichia coli chromosome, modulates the induction of plasmid-borne beta-lactamase genes by extracellular beta-lactam antibiotics. This suggests that the gene products AmpD and AmpE may function in the transduction of external signals. beta-Lactam antibiotics are analogs of cell wall components that can be released during cell wall morphogenesis of enterobacteria. The amp operon was studied to determine its importance in signal transduction during cell wall morphogenesis. The peptidoglycan compositions of amp mutants were determined by high-performance liquid chromatography and fast atom bombardment mass spectrometry. When a chromosomal or plasmid-borne copy of ampD was present, the amount of pentapeptide-containing muropeptides in the cell wall increased upon addition of the cell wall constituent diaminopimelic acid to the growth medium. These results suggest that beta-lactamase induction and modulation of the composition of the cell wall share elements of a regulatory circuit that involves AmpD. Escherichia coli requires AmpD to respond to extracellular signaling amino acids, such as diaminopimelic acid, and this signal transduction system may regulate peptidoglycan composition in response to cell wall turnover products.

Specific oxidative cleavage of carotenoids by VP14 of maize.

The plant growth regulator abscisic acid (ABA) is formed by the oxidative cleavage of an epoxy-carotenoid. The synthesis of other apocarotenoids, such as vitamin A in animals, may occur by a similar mechanism. In ABA biosynthesis, oxidative cleavage is the first committed reaction and is believed to be the key regulatory step. A new ABA-deficient mutant of maize has been identified and the corresponding gene, Vp14, has been cloned. The recombinant VP14 protein catalyzes the cleavage of 9-cis-epoxy-carotenoids to form C25 apo-aldehydes and xanthoxin, a precursor of ABA in higher plants.

Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation.

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

Hanging by a thread: invasion of legume plants by rhizobia.

Nitrogen-fixing nodules on plants such as alfalfa, pea and vetch arise from the root inner cortex and grow via a persistent meristem. Thus, these nodules are defined as indeterminate. The formation of functional indeterminate nodules requires that symbiotic bacteria, collectively called rhizobia, gain access to the interior of roots and root nodules via infection threads. Recent work has begun to elucidate the important functions of the root cell cytoskeleton in infection thread formation. It has also recently become apparent that rhizobial Nod factors and rhizobial exopolysaccharides play key roles in the initiation and elongation of infection threads.

Plant one-carbon metabolism and its engineering.

The metabolism of one-carbon (C1) units is vital to plants. It involves unique enzymes and takes place in four subcellular compartments. Plant C1 biochemistry has remained relatively unexplored, partly because of the low abundance or the lability of many of its enzymes and intermediates. Fortunately, DNA sequence databases now make it easier to characterize known C1 enzymes and to discover new ones, to identify pathways that might carry high C1 fluxes, and to use engineering to redirect C1 fluxes and to understand their control better.

Biosynthesis and shedding of murine lymphoma gangliosides.

Ganglioside biosynthesis and subsequent shedding are a potential mechanism contributing to tumor cell escape from the host immune response. As a first step in identifying active molecular species, structural characterization and quantification of the purified individual cellular and shed gangliosides of YAC-1 murine lymphoma cells were undertaken. These studies uncovered three striking changes in ganglioside metabolism in cells passaged in vivo, compared with cells cultured in vitro. (i) Marked inhibition of GalNAcGM1b synthesis: GM1b was present in an equal proportion to its biosynthetic product GalNAcGM1b in vitro, but was present in a 6-fold higher concentration in vivo. (ii) Marked inhibition of NeuGc synthesis: NeuGc, present in vitro in an up to 7-fold higher concentration than its biosynthetic precursor NeuAc, was decreased in relative concentration in vivo (1:1). (iii) Selectivity of shedding: ganglioside shedding in vitro was generalized with respect to both carbohydrate structure and ceramide structure (mainly d18:1-C24:1 and d18:1-C16:0), while in vivo, there was selective shedding of gangliosides containing NeuGc and the shorter chain fatty acid. The reduced synthesis of NeuGc and of GalNAcGM1b in vivo, and the selective shedding of more polar ganglioside species, also in vivo, show that the extracellular environment can markedly affect cellular ganglioside metabolism.

The effects of cholesterol oxidation products in sickle and normal red blood cell membranes.

The oxysterol content in normal and sickle red blood cell (RBC) membranes was assessed using thin-layer chromatography and capillary gas chromatography/mass spectrometry. Several more oxysterols were present in sickle RBCs compared to normal RBCs. Sickle RBC membranes had a higher concentration of 5 alpha,6 alpha-epoxycholesterol, 5 alpha-cholestane-3 beta,5,6 beta-triol, 7-ketocholesterol and 19-hydroxycholesterol than normal RBC membranes. The increased oxysterols in sickle RBC may be an effect of the increased oxidative stress which occurs in sickle RBC membranes. Physical characteristics of normal and sickle RBC membrane ghosts with and without inserted oxysterols were examined by Fourier transform infrared spectroscopy. The data are consistent with a greater sterol content in sickle cells compared to normal RBC membranes, and a possible oxysterol-cholesterol synergism.

Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels.

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.

S-Methylmethionine Conversion to Dimethylsulfoniopropionate: Evidence for an Unusual Transamination Reaction.

Leaves of Wollastonia biflora (L.) DC. synthesize the osmoprotectant 3-dimethylsulfoniopropionate (DMSP) from methionine via S-methylmethionine (SMM) and 3-dimethylsulfoniopropionaldehyde (DMSP-ald); no other intermediates have been detected. To test whether the amino group of SMM is lost by transamination or deamination, [methyl-2H3,15N]SMM was supplied to leaf discs, and 15N-labeling of amino acids was monitored, along with synthesis of [2H3]DMSP. After short incubations more 15N was incorporated into glutamate than into other amino acids, and the 15N abundance in glutamate exceeded that in the amide group of glutamine (Gln). This is more consistent with transamination than deamination, because deamination would be predicted to give greater labeling of Gln amide N due to reassimilation, via Gln synthetase, of the 15NH4+ released. This prediction was borne out by control experiments with 15NH4Cl. The transamination product of SMM, 4-dimethylsulfonio-2-oxobutyrate (DMSOB), is expected to be extremely unstable. This was confirmed by attempting to synthesize it enzymatically from SMM using L-amino acid oxidase or Gln transaminase K and from 4-methylthio-2-oxobutyrate using methionine S-methyltransferase. In each case, the reaction product decomposed rapidly, releasing dimethylsulfide. The conversion of SMM to DMSP-ald is therefore unlikely to involve a simple transamination that generates free DMSOB. Plausible alternatives are that DMSOB is channeled within a specialized transaminase-decarboxylase complex or that it exists only as the bound intermediate of a single enzyme catalyzing an unusual transamination-decarboxylation reaction.

Dependence of the murine antibody response to an anti-CDR2 VH peptide on immunogen formulation.

A peptide corresponding to the second complementarity determining region of the heavy chain (CDR2 VH) from a murine anti-CD4 monoclonal antibody, designated L202, was synthesized by solid phase methodology in a number of different antigenic forms, for the purpose of comparing the effectiveness of different adjuvant-carrier systems in the induction of a murine antibody response against the immunizing peptide and parent antibody molecule. Two of the synthetic constructs contained the palmitoyl and N-palmitoyl-cysteinyl-S-(2,3-palmitoyloxy)-propanediol (PAM3Cys) moieties, respectively, attached to the peptide amino terminus with the immunogen comprising liposomal formulations of each. A third immunogen consisted of the CDR2 VH peptide admixed with the PAM3Cys non-covalently and incorporated into liposomes (PAM3Cys + CDR2 VH). A fourth composition comprised the CDR2 VH peptide conjugated to KLH via the sulfhydryl of an added N terminal cysteine (KLH-CDR2 VH) and injected with Complete Freund's adjuvant (CFA). A fifth immunogen consisted of the CDR2 VH peptide synthesized on an octameric, branched polylysine core as a multiple antigenic peptide (MAP-CDR2 VH) injected in the presence of Freund's adjuvant. Groups of five mice were injected intramuscularly with each of these immunogens and bled at two week intervals. The highest anti-peptide gamma-immunoglobulin (IgG) responses (against uncoupled peptide by ELISA) after 56 days were obtained with mice receiving the PAM3Cys-CDR2 VH peptide. However, when screened against the CDR2 V(H) peptide present as the MAP derivative by ELISA, IgG raised against the cognate MAP-CDR2 peptide was much more reactive than IgG raised against the liposomal PAM3Cys-CDR2 VH immunogen. In either case, IgG raised against the KLH-CDR2VH conjugate was poorly reactive. These differences in reactivity to the two forms of the CDR2 VH peptide by ELISA did not correspond to major differences in reactivities to the intact L202 Ab by ELISA. Although the IgG against the MAP immunogen was slightly more reactive than the other antisera against the l202 Ab, all titers were less than 1:100. These data illustrate some limitations of using anti-peptide responses as indicators of potential reactivity against the native protein, but suggest that alternate formulations including lipoidal peptides are more effective than corresponding KLH-peptide conjugates in eliciting Ab responses against poorly immunogenic epitopes.

Caprine mucopolysaccharidosis-IIID: clinical, biochemical, morphological and immunohistochemical characteristics.

Several animal models have been developed for the mucopolysaccharidoses (MPSs), a group of lysosomal storage disorders caused by lysosomal hydrolase deficiencies that disrupt the catabolism of glycosaminoglycans (GAG). Among the MPS, the MPS-III (Sanfilippo) syndromes lacked an animal counterpart until recently. In this investigation of caprine MPS-IIID, the clinical, biochemical, morphological, and immunohistochemical studies revealed severe and mild phenotypes like those observed in human MPS III syndromes. Both forms of caprine MPS IIID result from a nonsense mutation and consequent deficiency of lysosomal N-acetylglucosamine 6-sulfatase (G6S) activity and are associated with tissue storage and urinary excretion of heparan sulfate (HS). Using special stains, immunohistochemistry, and electron microscopy, secondary lysosomes filled with GAG were identified in most tissues from affected goats. Primary neuronal accumulation of HS and the secondary storage of gangliosides were observed in the central nervous system (CNS) of these animals. In addition, morphological changes in the CNS such as neuritic expansions and other neuronal alterations that may have functional significance were also seen. The spectrum of lesions was greater in the severe form of caprine MPS IIID and included mild cartilaginous, bony, and corneal lesions. The more pronounced neurological deficits in the severe form were partly related to a greater extent of CNS dysmyelination. These findings demonstrate that caprine MPS IIID is a suitable animal model for the investigation of therapeutic strategies for MPS III syndromes.

The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein.

The inhibitor protein (IP) that inactivates spinach leaf NADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14-3-3 protein family based on three lines of evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14-3-3 proteins. Second, an authentic member of the 14-3-3 family, recombinant Arabidopsis GF14omega, caused inactivation of phospho-NR in a magnesium-dependent manner identical to IP. Third, an anti-GF14 monoclonal antibody cross-reacted with IP and anti-IP monoclonal antibodies cross-reacted with GF14omega.

Gastric distention by balloon and test-meal intake in obese and lean subjects.

A study was conducted to determine the effects of various levels of gastric distension on spontaneous meal intake. A balloon was inserted into the stomach of four lean and four obese subjects before consumption of a lunch meal. On different days the balloon was filled with 0, 200, 400, 600, and 800 mL water in a random sequence. As balloon volume increased, food intake decreased, with a balloon volume of greater than or equal to 400 mL significantly reducing intake (p less than 0.01). There was no significant difference between lean and obese subjects. Because gastric emptying rate was not significantly slowed by a volume of 800 mL, between emptying was probably not a factor in inducing satiety. Discomfort was probably also not a factor because it was experienced by only two lean subjects at 800 mL. Most likely gastric distension itself triggered satiety signals.

Gastric balloon to treat obesity: a double-blind study in nondieting subjects.

To determine its efficacy and safety in treating obesity, a silicone-rubber balloon was passed into the stomach of 10 nondieting, obese subjects. In a counterbalanced sequence, the balloon was inflated with 400 mL for 1 mo and deflated for 1 mo. Lower intakes of solid and liquid test meals (NS), significantly slower gastric emptying, and concomitant changes in glucose, insulin, glucagon, and cholecystokinin concentrations consistent with slower emptying resulted during balloon inflation. After balloon inflation, one small gastric ulcer developed, which subsequently healed. Significant weight loss occurred during the second and third week of the inflation period (F[1,9] = 5.0, p less than 0.05). However, the weight loss was small and the significant effect did not continue through the fourth week.

Gastric capacity, gastric emptying, and test-meal intake in normal and bulimic women.

The role of the stomach in regulating appetite in bulimia nervosa was examined. Subjects were nine normal and nine bulimic women of similar age, height, and weight. Gastric capacity was estimated by filling a balloon in the stomach. The mean stomach capacity of bulimic subjects was significantly larger than that of normal subjects, as revealed by the larger balloon volume tolerated (P less than 0.01) and by the larger volume needed to produce a 5 cm H2O increase in intragastric pressure (P = 0.07). The intake of a liquid meal was also significantly larger for the bulimic subjects. Gastric-emptying rate of a liquid meal was significantly delayed in the bulimic subjects during the initial 5-15 min. In all subjects, test-meal intake correlated significantly with gastric capacity (r = 0.53). In the bulimic subjects, self-reported binge intake (J) also correlated significantly with gastric capacity (r = 0.75). Binge eating in bulimic subjects may enlarge gastric capacity, which could then promote even larger binges through positive feedback.

A comparison of the effects of buspirone and diazepam on plasma corticosterone levels in rat.

The effect of the anxiolytic agents, buspirone and diazepam, on the hypothalamic-pituitary-adrenal axis (HPAA), indicated by changes in the concentration of corticosterone (CS) in plasma, were studied 1/2, 1, 2, 4, 6, 8 and 24 hr after administration of the drug (i.p.). Samples of plasma were collected in the mid-morning (0930-1130 hr) when activity in the hypothalamic-pituitary-adrenal axis in the rat and control levels of corticosterone were low and were repeated in the afternoon (1400-1600 hr) when activity in the hypothalamic-pituitary-arenal axis and levels of corticosterone were higher. At small doses (1 mg/kg) buspirone had a greater facilitating effect on the hypothalamic-pituitary-adrenal axis than did diazepam. In addition, buspirone had a greater maximum facilitatory effect (477%) on levels of corticosterone than diazepam (345%). However, buspirone (ED50 = 8.6 mumol/kg) and diazepam (ED50 = 8.7 mumol/kg) were equipotent. Administration of 1 mg/kg of buspirone in the morning increased the combined 1/2 and 1 hr circulating levels of corticosterone 75% above control levels. Diazepam, at 1 mg/kg, did not produce any significant changes in levels of corticosterone. Large doses (10 mg/kg) of buspirone increased morning levels of corticosterone by 328% and diazepam increased levels of corticosterone by 265%. During the afternoon small doses of buspirone or diazepam did not significantly alter levels of corticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Betaine aldehyde, betaine, and choline levels in rat livers during ethanol metabolism.

Betaine aldehyde levels were determined in rat livers following 4 weeks of ethanol feeding, employing the Lieber-De Carli liquid diet. The results showed that the levels of betaine aldehyde are unaffected by alcohol feeding to rats. These levels in both experimental and control animals were found to be quite low, 5.5 nmol/g liver. Betaine aldehyde levels have not been determined previously in mammalian liver because of methodological difficulties. This investigation employed fast atom bombardment-mass spectroscopy to determine the levels of betaine aldehyde, betaine, and choline. The decrease in betaine levels following ethanol administration confirmed the results of other investigators. Choline levels determined during this investigation were lower than previously reported. The reason for starting this investigation was the fact that the enzyme that catalyzes betaine aldehyde dehydrogenation to betaine, which is distributed in both mitochondria and the cytoplasm, was found to also metabolize acetaldehyde with K(m) and V(max) values lower than those for betaine aldehyde. Thus, it appeared likely that the metabolism of acetaldehyde during ethanol metabolism might inhibit betaine aldehyde conversion to betaine and thereby result in decreased betaine levels (Barak et al., Alcohol 13: 395-398, 1996). The fact that betaine aldehyde levels in alcohol-fed animals were similar to those in controls demonstrates that competition between acetaldehyde and betaine aldehyde for the same enzyme does not occur. This complete lack of competition suggests that betaine aldehyde dehydrogenase in the mitochondrial matrix may totally metabolize betaine aldehyde to betaine without any involvement of cytoplasmic betaine aldehyde dehydrogenase.