Emergence from general anaesthesia and evolution of delirium signs in the post-anaesthesia care unit.

BACKGROUND: Emergence from anaesthesia is often accompanied by signs of delirium, including fluctuating mental status and inattention. The evolution of these signs of delirium requires investigation since delirium in the post-anaesthesia care unit (PACU) may be associated with worse outcomes. METHODS: Adult patients emerging from anaesthesia were assessed for agitated emergence in the operating room using the Richmond Agitation-Sedation Scale (RASS). The Confusion Assessment Method for the Intensive Care Unit was then used to evaluate delirium signs at PACU admission and during PACU stay at 30 min, 1 h, and discharge. Signs consistent with delirium were classified as hyperactive vs hypoactive based upon a positive CAM-ICU assessment and the concomitant RASS score. Multivariable logistic regression was utilized to assess potential risk factors for delirium during PACU stay including age, American Society of Anesthesiologists classification, and opioid and benzodiazepine exposure. RESULTS: Among 400 patients enrolled, 19% had agitated emergence. Delirium signs were present at PACU admission, 30 min, 1 h, and PACU discharge in 124 (31%), 59 (15%), 32 (8%), and 15 (4%) patients, respectively. In patients with delirium signs, hypoactive signs were present in 56% at PACU admission and in 92% during PACU stay. Perioperative opioids were associated with delirium signs during PACU stay (P=0.02). CONCLUSIONS: A significant proportion of patients develop delirium signs in the immediate postoperative period, primarily manifesting with a hypoactive subtype. These signs often persist to PACU discharge, suggesting the need for structured delirium monitoring in the PACU to identify patients potentially at risk for worse outcomes in the postoperative period.

Three-dimensional imaging and analysis of the surface of hair fibres using scanning electron microscopy.

Cuticle scales are the most obvious feature of a hair fibre's outer surface and much research has focused on assessing the influence of surface topography on the associated hair fibre's properties. However, much of the research has either been qualitative or, if quantitative, employed relatively laborious analytical techniques to establish the necessary statistical robustness. In this study, we report on the application of a 3D image analysis package capable of producing 3D data from multiple 2D scanning electron microscope (SEM) images of hair fibres. Analysis of the surface profile can be carried out quickly and accurately, enabling quantification of the scale structure. To validate the novel technique and ensure that the scale heights measured were indeed accurate and reproducible, extensive calibration of the SEM and the 3d software has been performed. In addition, scale heights on a single hair have been determined by using atomic force microscopy (AFM) and the results compared with analogous data produced from the same scale edges using the 3D image analysis technique. The data obtained indicate that a relatively quick, accurate and viable method to determine scale height in keratin fibres has been established. A further 3D-SEM analysis method has been developed which allows individual scales to be monitored and for the cuticular scale height to be quantified after repeated 'smoothing' treatments.

Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association.

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.

alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.

Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction.

The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.

Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.

Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor.

The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.

Rab3A triggers the acrosome reaction in permeabilized human spermatozoa.

The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.

Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation.

It has long been known that seminal plasma contains factors that influence the fertilizing capacity of spermatozoa in many different ways. However, little is understood of the biochemical cascades triggered when spermatozoa and seminal plasma interact. In this study, we examined how incubation with seminal plasma affected protein tyrosine phosphorylation in human spermatozoa. Increased protein tyrosine phosphorylation is a hallmark of sperm capacitation in several mammalian species, including human. Seminal plasma blocks protein tyrosine phosphorylation when added to washed, non-capacitated spermatozoa. Removal of seminal plasma and incubation in capacitating medium led to partial recovery of the tyrosine phosphorylation cascade. Addition of seminal plasma to a suspension of spermatozoa previously incubated for 5 h under capacitating conditions decreased the level of tyrosine phosphorylation on all proteins in a dose-dependent manner. In this case, the phosphotyrosine signal did not increase upon removal of seminal plasma followed by overnight incubation in fresh capacitating media, indicating that removal of seminal plasma was necessary but not sufficient for protein tyrosine phosphorylation to occur. These results indicate that human seminal plasma contains factors that influence the tyrosine phosphorylation status of human spermatozoa.

Activation of mouse sperm phosphatidylinositol-4,5 bisphosphate-phospholipase C by zona pellucida is modulated by tyrosine phosphorylation.

Many cellular responses to the occupancy of membrane receptors include the hydrolysis of phosphatidylinositol-4,5 bisphosphate (PIP2) by phospholipase C (PLC) and the subsequent generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). In the gamete interaction system, sperm respond to binding to the egg's extracellular matrix, the zona pellucida (zp), by exocytosis of the acrosome in a process known as the acrosome reaction (AR). Under physiological conditions, zp binding stimulates ARs only after sperm have undergone a final maturation phase, known as capacitation. One of the zp glycoproteins, ZP3, serves as the ligand for sperm plasma membrane receptors and as the trigger for this regulated exocytosis. Both phosphoinositide-linked and tyrosine kinase-mediated pathways participate in the signalling cascade triggered by sperm-zp interaction. This paper reports that stimulation with solubilized zp increased PIP2-PLC enzymatic activity from mouse sperm. ZP3 is the zp component responsible for this stimulation. The effect was abolished by tyrphostin, suggesting that zp activation of PLC was mediated by tyrosine phosphorylation and that gamma was the PLC isoform involved. We show the presence and distribution of PLC gamma 1 in mouse sperm. Immunostaining studies indicate that PLC gamma 1 is restricted to the sperm head. Sperm capacitation induced translocation of PLC gamma 1 from the soluble to the particulate fraction. These data suggest that PLC gamma 1 constitutes a component in the cascade that couples sperm binding to the egg's extracellular matrix with acrosomal exocytosis, a regulated secretory response upon which fertilization depends absolutely.

LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm: evidence that the antigen recognized is not hexokinase.

In the mouse, a 95 kD sperm protein has been identified as a putative receptor for the zona pellucida glycoprotein ZP3. The 95 kD sperm protein is a tyrosine kinase substrate, with phosphorylation on tyrosine stimulated upon zona pellucida binding. The latter finding is observed not only in live cells but also in isolated sperm membranes and in an electroeluted 95 kD protein. Stimulation of 95 kD protein tyrosine phosphorylation by zona pellucida is completely abolished by tyrosine kinase inhibitors, which effectively inhibit the sperm acrosome reaction. Since receptor oligomerization by ZP3 is essential for acrosome reaction triggering, we hypothesized that application of an external crosslinking agent will lead to the acrosome reaction, even in the absence of natural ligand ZP3. Here, we report the generation of a mouse monoclonal antibody (mAb) raised against the 95 kD protein. This antibody, termed LL95, mimics the bioactivities of ZP3 in inhibiting sperm-zona binding and inducing the acrosome reaction. The latter depends on receptor oligomerization. Immunolocalization revealed that the LL95 antigen is restricted to the head surface in the acrosomal region of live sperm. Thus, LL95 fulfills several criteria predicted for an antibody that recognizes a sperm receptor for the zona pellucida. Recently, it was reported that the amino acid sequence of the 95 kD protein we described corresponds to a mouse hepatoma hexokinase (Kalab et al., 1994: J Biol Chem 269:3810-3817). Although both hexokinase and LL95 antigen migrate at 95 kD in nonreducing gels, we show here that LL95 does not recognize hexokinase. Identification of different proteins is clear where hexokinase is a 116 kD protein and LL95 recognizes sperm proteins of 110 and 130 kD. Moreover, mAb anti-phosphotyrosine immunoprecipitates LL95 antigen under conditions where hexokinase is absent. Use of anti-hexokinase antibodies in gamete interaction assays failed to demonstrate any effect on either sperm-zona binding or acrosome reaction triggering. Finally, antihexokinase antibodies bind to a sperm tail antigen, thus direct involvement of hexokinase in gamete interaction seems improbable.

Isobutylmethylxanthine and other classical cyclic nucleotide phosphodiesterase inhibitors affect cAMP-dependent protein kinase activity.

The effect of 17 inhibitors of cyclic nucleotide phosphodiesterases (PDEs) was assayed on cAMP binding activity of Mucor rouxii protein kinase A (PKA), on PKA activity in the absence of cAMP and on free catalytic subunit (C) activity. Isobutylmethylxanthine (IBMX), SQ 20,009 and cilostamide, at 0.2 mM, behaved as partial agonists of cAMP since they inhibited binding of 0.15 microM [3H]cAMP to the regulatory subunit (R), stimulated slightly PKA activity in the absence of cAMP and did not modify C activity. Amrinone at 0.2 mM inhibited C activity competitively towards ATP. These four compounds displayed the same effects when assayed on eukaryotic protein kinase A types I (PKI) and II (PKII). The combined effect of IBMX and cAMP was analysed on Mucor PKA. Under dissociating conditions (+ 0.5 M NaCl) IBMX antagonized activation by low concentrations of cAMP, while in the absence of NaCl, IBMX potentiated the stimulating activity of cAMP.

Antimicrobial constituents of Gomphrena martiana and Gomphrena boliviana.

The antimicrobial activity of extracts and constituents of Gomphrena martiana and Gomphrena boliviana (Amaranthaceae) were determined in order to identify the compounds responsible for the folk-medicinal use of these plants. Each extract was evaluated against 20 microorganisms, including Gram-positive and Gram-negative bacteria, spore-forming Gram-positive bacteria, an acid-fast bacterium, a fungus and two yeasts. Fractionation of each petroleum ether (PE) extract yielded five 5,6,7-trisubstituted flavones that were separately tested showing high activity against M. phlei (minimum inhibitory concentration (MIC) 15, 20 and 75 micrograms/ml) approaching that of commercial bactericides. Other natural and synthetic flavonoids with diverse structures were also tested to define structure-activity relationships. Each EtOH extract was subsequently fractionated and monitored by bioassays leading to isorhamnetin 3-O-beta-robinobioside (MIC 50 micrograms/ml) in both instances. This glycoside is reported here for the first time in G. boliviana.

cAMP analogs and selective inhibitors used to study low Km Mucor rouxii cAMP phosphodiesterase.

1. The sensitivity of partially purified low Km phosphodiesterase (PDE) from Mucor rouxii to pharmacological agents and cAMP analogs was studied. The IC50 obtained were compared with those reported for PDEs from higher eukaryotes. 2. The best inhibitors of the hydrolysis of 1 microM cAMP were SQ 65.442 (IC50 c 10 microM), dipyridamol and CI 930. cGMP was not an inhibitor (IC50 greater than 1000 microM). 3. The cAMP analogs were tested as inhibitors of the hydrolysis of 0.1 microM cAMP. 8-Aminohexylamino cAMP was the best inhibitor with an IC50 of c 1 microM. 4. A sedimentation profile of Mucor PDE was assayed in the presence of several pharmacological inhibitors and cAMP analogs. No isoforms with different sensitivity towards the inhibitors were detected. Forms with slightly different behaviour towards some cAMP analogs were observed.

Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii.

A partially purified preparation (200-fold) of cAMP phosphodiesterase (PDE) was obtained from Mucor rouxii grown and extracted under conditions minimizing endogenous proteolysis. Four purification steps were applied: batch DEAE-Sepharose, DEAE-Sepharose chromatography, Sephadex G-150 super-fine gel filtration and sucrose gradient centrifugation. The final PDE preparation was activatable by cAMP-dependent phosphorylation and controlled trypsin treatment. A careful correlation of protein patterns with PDE activity was done throughout the whole procedure by analyzing the active fractions of each step by mini-polyacrylamide non-denaturing gel electrophoresis. The final preparation displayed four major protein bands, none of which corresponded to PDE, although PDE activity comigrated with two of them. Some properties of this preparation were studied. Vmax increased around 10-15 fold by activation of PDE by phosphorylation or proteolysis; Km values were unaffected. PDE had Stokes radius of 3.5 nm, sedimentation coefficient of 4.3 S and molecular weight of 70,000 daltons. The treatment of sucrose gradient fractions with [gamma-32P] ATP and cAMP-dependent protein kinase catalytic subunit and further analysis through minigels showed that none of the visible bands was phosphorylated, and that among the four phosphorylated bands there was one that cosedimented and comigrated with PDE activity. Trypsin treatment of the phosphorylated samples removed the label but did not modify the staining pattern.