Laminar airflow decreases microbial air contamination compared with turbulent ventilated operating theatres during live total joint arthroplasty: a nationwide survey.

BACKGROUND: Preventing surgical site infections and prosthetic joint infections is crucial for patient safety after total joint arthroplasty. Microbial air contamination has been suggested as a risk factor. Therefore, the ventilation system that will reduce air contamination most effectively in operating theatres (OTs) has been discussed. AIM: To determine whether laminar airflow (LAF) ventilation is superior to turbulent airflow (TAF) ventilation by looking at the colony forming units (cfu) count during live total hip and knee arthroplasties. Furthermore, to explore whether the number of OT personnel, door and cabinet lock openings and technical parameters of the ventilation systems have an impact on the number of cfu. METHODS: Active air sampling and passive sedimented bacterial load were performed in 17 OTs, equipped with either LAF or TAF ventilation, during 51 live surgeries while observations were noted. FINDINGS: LAF OTs reduced cfu counts compared with TAF OTs during live surgery (P<0.001). All LAF OTs provided ultraclean air whereas TAF had nine procedures exceeding the threshold of 10 cfu/m3. Door and cabinet lock openings and number of personnel did not influence the cfu count, while it decreased with increasing volume and total air change per hour (P<0.05). CONCLUSION: All LAF OTs had cfu counts within recommendations and provided lower cfu counts compared with TAF OTs. The number of OT personnel and total openings did not have an influence on cfu counts. Increased volume of the OT and total air change per hour showed a decrease in active cfu counts.

Functional roles of conserved transmembrane prolines in the human VPAC(1) receptor.

The importance of three conserved transmembrane prolines of the human vasoactive intestinal polypeptide (VPAC)(1) receptor was examined by single alanine substitution. P266A, P300A and P348A reduced the expression level, but maintained the binding to VIP. P266A showed decreased ability to stimulate cAMP, while P300A and P348A displayed an increased potency in cAMP production combined with a high sensitivity towards GTP compared to the wild type receptor. In addition, substitutions of two conserved leucines located in position -2 and +1 from P348 were investigated. L346A and L349A reduced the receptor expression, influenced the G protein coupling and decreased the receptor activity. These observations, which are the first on conserved transmembrane prolines within this family of receptors, indicate that these residues are important for receptor expression, G protein coupling and receptor activity.

Pituitary adenylate cyclase-activating polypeptide induces period1 and period2 gene expression in the rat suprachiasmatic nucleus during late night.

The suprachiasmatic nucleus generates circadian rhythms which are synchronized to the environmental light-dark cycle via the retinohypothalamic tract. Pituitary adenylate cyclase-activating polypeptide and glutamate, two neurotransmitters co-stored in the retinohypothalamic tract of the rat, are able to phase shift the endogenous rhythm similar to light. The "clock genes" period1 (per1) and per2, which show circadian oscillation within the suprachiasmatic nucleus, have been attributed a role in light-induced resetting of the mammalian circadian clock due to rapid induction of the period (per) genes after light stimulation at night. Using a rat in vitro brain slice model, we demonstrate by quantitative in situ hybridization histochemistry that the diurnal alteration in expression of both per genes in the suprachiasmatic nucleus was retained in vitro. In the model, we examined the effects of pituitary adenylate cyclase-activating polypeptide and glutamate alone and in combination on per1 and per2 gene expression at late subjective night (circadian time 19). Glutamate administration (10(-3)M) induced both per1 and per2 gene expression in the suprachiasmatic nucleus of the brain slice within 1h. The per gene responses were similar to the induction of gene expression observed after light stimulation in vivo at late night. Pituitary adenylate cyclase-activating polypeptide (10(-6)M) administered alone had no effect on the per gene expression, but when pituitary adenylate cyclase-activating polypeptide in micromolar concentration was applied before glutamate, the neuropeptide blocked the glutamate-induced per1 and per2 gene expression in the suprachiasmatic nucleus. In contrast to the lack of effect of pituitary adenylate cyclase-activating polypeptide itself in micromolar concentration, pituitary adenylate cyclase-activating polypeptide (10(-9)M) induced both per1 and per2 gene expression, an effect which was not augmented by co-application of glutamate. Our results provide the molecular substrate for the previous electrophysiological findings that pituitary adenylate cyclase-activating polypeptide in high concentration is able to block glutamate-induced phase advance at late night, and that the peptide in low concentration can induce a phase advance similar to light and glutamate.

Characterization of a G protein coupling "YL" motif of the human VPAC1 receptor, equivalent to the first two amino acids in the "DRY" motif of the rhodopsin family.

The conserved residues Y239 and L240 of human VPAC1 receptor are predicted to be at the same location as the asparagine and arginine in the "DRY" motif in the Rhodopsin family of G protein-coupled receptors. By comparing vasoactive intestinal peptide (VIP) binding with or without the presence of GTP-gamma-S, it was found that the deltadelta G(o) for the endogenous G-protein coupling was 1.5 kJ/mol, 0.95 kJ/mol, and 3.4 kJ/mol for theY239A, L240A, and wild-type receptor, respectively. VIP-induced cAMP production in whole cells support the results of the binding studies, as Y239A had a moderate and L240A a pronounced impaired ability to produce cAMP. The mutants had a minor influence on the intrinsic "low affinity to high affinity equilibrium," suggesting that the dominating effect of these mutants is a perturbation of the G protein-binding site. Thus, the highly diverged chemical properties of the hydrophobic "YL" motif and charged "DR(Y)" motif could be a crucial difference between the Secretin Receptor Family and the Rhodopsin Family with respect to receptor activation and G-protein coupling.

Role of second extracellular loop in the function of human vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide receptor 1 (hVPAC1R).

To elucidate the functional role of the second extracellular loop of human vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide (VIP/PACAP) receptor (hVPAC1R), surface expression, ligand binding, and receptor activation were analyzed. Amino acids in the entire second extracellular loop were individually substituted by alanine by site-directed mutagenesis. The mutant and wild-type receptors were transiently expressed in HEK293 cells and purified cell membranes were tested for the ability to bind VIP, while the receptor activity was measured as potency of cAMP production analysed on intact cells. Surface expression of the substituted conserved residues, W286A, I289A, W294A, and W295A, was evidently decreased to 20-30% compared to the wild-type expression. W286A also showed an significantly reduced potency of cAMP production. Substituted residues as F280A, E281A, and G284A showed a significant reduction in the potency of stimulated cAMP production amounting to 8-46-fold, compared to the wild-type with unaffected surface expression and VIP binding. These results indicate that some residues in the second extracellular loop of the human VPAC1R participate in the active mechanism of a ligand-mediated response without being directly involved in the binding of VIP.

A role for upstream RNA structure in facilitating the catalytic fold of the genomic hepatitis delta virus ribozyme.

Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HDV contain a ribozyme that undergoes cis-cleavage, thereby processing the transcript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have attenuated self-cleavage. Structure mapping and site-directed mutagenesis revealed an inhibitory stretch consisting of upstream nucleotides -24 to -15 that forms a long-range pairing, termed Alt 1, with the 3' strand of P2 (P2(3')) located at the very 3'-end of the ribozyme. Two other alternative pairings were found, Alt 2, which involves upstream nucleotide-ribozyme interactions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleavage was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the -24/-15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased to 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides -54 to -18, termed P(-1)/L(-1), which sequesters the majority of the -24/-15 inhibitory stretch in cis. This hairpin is followed by a stretch of single-stranded pyrimidine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, and that the J(-1/1) stretch is conserved among antigenomic HDV isolates. Lastly, the secondary structure of the Alt 1-containing ribozyme provides insight into possible folding intermediates of the ribozyme.

Proposed arrangement of the seven transmembrane helices in the secretin receptor family.

An arrangement of the seven transmembrane alpha-helices of the G protein-coupled Secretin receptor family is proposed. The helices of 27 homologous receptor sequences were plotted as helical wheels. The solvent inaccessible portion of each helix was used to assign relative orientations. They were arranged according to two criteria: 1) conserved, hydrophilic residues and aligned positions with restricted volume changes face the other helices and 2) aligned positions with low identity and large volume change face the lipid. The positive inside rule confirms the assumption that loops connecting transmembrane helices I-II, III-IV, V-VI and the C-terminal part of the receptors are intracellular. Our model approach was tested using the Bacteriorhodopsin family. The use of volume changes at each position in the transmembrane helix was crucial for the good correlation of the orientation of the helices using the model approach and the structure of bacteriorhodopsin solved by electron microscopy [Grigorieff N, Ceska TA, Downing KH, Baldwin JM, and Henderson R (1996) J Mol Biol 259 393-421]. The tests of our modelling approach showed that six helices were within a 15 degrees derivation in the orientation and five helices were within a horizontal derivation of two residues. The largest orientational derivations of a helix were 40 degrees and the largest horizontal displacement was four residues. A long stretch of side chains predicted to possess low resistance to movement in helix V of the Secretin receptor family suggests an involvement in receptor activation. Comparison of the Secretin receptor family and the larger G protein-coupled Rhodopsin family showed many similarities, despite the lack of obvious sequence identity.

Importance of conserved cysteines in the extracellular loops of human PACAP/VIP1 receptor for ligand binding and stimulation of cAMP production.

The importance of two highly conserved cysteines in the human pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide 1 (VIP1) receptor was examined. Using site-directed mutagenesis, each Cys residue was converted into Ala or Ser. The mutant and wildtype genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215Ala/Ser and Cys285Ala/Ser showed at least a tenfold decrease in binding affinity and receptor potency when compared to the wildtype. In contradiction to the wildtype receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilizing the receptor in the correct conformation for ligand binding and activation.

A disulfide bond between conserved cysteines in the extracellular loops of the human VIP receptor is required for binding and activation.

The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor I (hVIPR 1) was examined. By site-directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild-type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215-Ala/Ser and Cys285-Ala/Ser showed at least a 10-fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild-type receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilising the receptor in the correct conformation for ligand binding and activation.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in the mesencephalic trigeminal nucleus of the rat after transsection of the masseteric nerve.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the VIP (vasoactive intestinal polypeptide) family of peptides, has been demonstrated in neurons of the sensory system. PACAP expression of these neurons is sensitive to nerve damages such as nerve crush or axotomy. In the present study, PACAP expression in the mesencephalic trigeminal nucleus of the rat was examined after transsection of the main trunk of the masseteric nerve. The primary sensory neurons of the nucleus are considered to have purely proprioceptive functions. By quantitative in situ hybridization using a PACAP [35S]cRNA probe, an increase in PACAP mRNA was observed on the side ipsilateral to transsection already after 3 h and the expression reached a peak 24 h after surgery after which the levels gradually decreased during the next 14 days. A low and constant expression of PACAP mRNA could be seen on the side contralateral to transsection. PACAP immunoreactivity was demonstrated on the ipsilateral side after 18 h, using a specific monoclonal PACAP antibody. Co-existence of PACAP with NPY and galanin was demonstrated 7 days after transsection. Analysis of the masseteric nerve by radioimmunoassay on transsected and normal nerve stumps revealed an increase of PACAP-38 immunoreactivity in the nerve proximal to the transsection compared to the normal side (15.3 vs. 6.1 pmol/g wt). The results suggest that PACAP has a role in the early phase of adaptation to nerve injury in the proprioceptive neurons.

Assignment of human elongation factor 1alpha genes: EEF1A maps to chromosome 6q14 and EEF1A2 to 20q13.3.

The human elongation factor 1alpha gene family consists of at least 2 actively transcribed genes, EEF1A and EEF1A2, and more than 18 homologous loci. EEF1A2 is expressed in a tissue-specific manner, whereas EEF1A is expressed ubiquitously, and both of them can function in translation. An EEF1A cDNA probe has previously been shown to cross-hybridize with several human chromosomes, but the location of the functional gene has not been established. We have mapped the functional EEF1A gene to 6q14 by combined fluorescence in situ hybridization (FISH) and PCR analysis of a somatic cell hybrid panel and mapped EEF1A2 to 20q13.3 by FISH. In addition, the 11 strongest cross-hybridizing loci (EEF1AL2-EEF1AL13) were mapped by FISH to 12p12, 9q34, 7p15-p21, 19q13, 3q26-q27, 7q33-q35, 1p13-p22, 2q12-q14, 5p12-q11, 1q31-q32, and Xq21.

Tissue-dependent variation in the expression of elongation factor-1 alpha isoforms: isolation and characterisation of a cDNA encoding a novel variant of human elongation-factor 1 alpha.

A novel isoform of human elongation factor-1 alpha (EF-1 alpha 2) has been characterised. It shows a high similarity to other EF-1 alpha proteins, especially to a rat EF-1 alpha variant and it has all the characteristics of a functional EF-1 alpha protein. The pattern of expression of both EF-1 alpha 2 and EF-1 alpha was analysed in different human tissues. This showed that the two proteins were differentially expressed, EF-1 alpha 2 was expressed in brain, heart, skeletal muscle and in the transformed cell lines AMA and K14, but was undetectable in other tissues and in both primary and transformed human fibroblasts. EF-1 alpha was expressed in brain, placenta, lung, liver, kidney, pancreas and in all the cell lines that we have analysed but barely detectable in heart and skeletal muscle.

Development of efficient suicide mechanisms for biological containment of bacteria.

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.