Automated sampling and data processing derived from biomimetic membranes.

Recent advances in biomimetic membrane systems have resulted in an increase in membrane lifetimes from hours to days and months. Long-lived membrane systems demand the development of both new automated monitoring equipment capable of measuring electrophysiological membrane characteristics and new data processing software to analyze and organize the large amounts of data generated. In this work, we developed an automated instrumental voltage clamp solution based on a custom-designed software controller application (the WaveManager), which enables automated on-line voltage clamp data acquisition applicable to long-time series experiments. We designed another software program for off-line data processing. The automation of the on-line voltage clamp data acquisition and off-line processing was furthermore integrated with a searchable database (DiscoverySheet) for efficient data management. The combined solution provides a cost efficient and fast way to acquire, process and administrate large amounts of voltage clamp data that may be too laborious and time consuming to handle manually.

Recombination in Mycoplasma hominis.

Mycoplasma hominis has been previously described as a heterogeneous species, and in the present study intraspecies diversity of 20 M. hominis isolates from different individuals was analyzed using parts of the unlinked gyrase B (gyrB), elongation factor Tu (tuf), SRalpha homolog (ftsY), hitB-hitL, excinuclease ABC subunit A (uvrA) and glyceraldehyde-3-phosphate dehydrogenase (gap) genes. The level of variability of these M. hominis genes was low compared with the housekeeping genes from Helicobacter pylori and Neisseria meningitidis, but only few M. hominis isolates had identical sequences in all genes indicating the presence of recombination. In order to test for intergenic recombination, phylogenetic trees were reconstructed for each of the genes but no well-supported bifurcating phylogenetic trees could be obtained. The genes were tested for intragenic recombination using the correlation between linkage disequilibrium and distance between the segregating sites, by the homoplasy ratio (H ratio), and by compatibility matrices. The gap gene showed well-supported evidence for high levels of recombination, whereas recombination was less frequent and not significant within the other genes. The analysis revealed intergenic and intragenic recombination in M. hominis and this may explain the high intraspecies variability. The results obtained in the present study may be of importance for future population studies of Mycoplasma species.

Molecular design of Mycoplasma hominis Vaa adhesin.

The variable adherence-associated (Vaa) adhesin of the opportunistic human pathogen Mycoplasma hominis is a surface-exposed, membrane-associated protein involved in the attachment of the bacterium to host cells. The molecular masses of recombinant 1 and 2 cassette forms of the protein determined by a light-scattering (LS) method were 23.9 kD and 36.5 kD, respectively, and corresponded to their monomeric forms. Circular dichroism (CD) spectroscopy of the full-length forms indicated that the Vaa protein has an alpha-helical content of approximately 80%. Sequence analysis indicates the presence of coiled-coil domains in both the conserved N-terminal and antigenic variable C-terminal part of the Vaa adhesin. Experimental results obtained with recombinant proteins corresponding to the N- or C-terminal parts of the shortest one-cassette form of the protein were consistent with the hypothesis of two distinct coiled-coil regions. The one-cassette Vaa monomer appears to be an elongated protein with a axial shape ratio of 1:10. Analysis of a two-cassette Vaa type reveals a similar axial shape ratio. The results are interpreted in terms of the topological organization of the Vaa protein indicating the localization of the adherence-mediating structure.

Synthesis of pyrrolidinone PNA: a novel conformationally restricted PNA analogue.

To preorganize PNA for duplex formation, a new cyclic pyrrolidinone PNA analogue has been designed. In this analogue the aminoethylglycine backbone and the methylenecarbonyl linker are connected, introducing two chiral centers compared to PNA. The four stereoisomers of the adenine analogue were synthesized, and the hybridization properties of PNA decamers containing one analogue were measured against complementary DNA, RNA, and PNA strands. The (3S,5R) isomer was shown to have the highest affinity toward RNA, and to recognize RNA and PNA better than DNA. The (3S,5R) isomer was used to prepare a fully modified decamer which bound to rU10 with only a small decrease in Tm (delta Tm/mod = 1 degree C) relative to aminoethylglycine PNA.

A highly selective CC chemokine receptor (CCR)8 antagonist encoded by the poxvirus molluscum contagiosum.

The MC148 CC chemokine from the human poxvirus molluscum contagiosum (MCV) was probed in parallel with viral macrophage inflammatory protein (vMIP)-II encoded by human herpesvirus 8 (HHV8) in 16 classified human chemokine receptors. In competition binding using radiolabeled endogenous chemokines as well as radiolabeled MC148, MC148 bound with high affinity only to CCR8. In calcium mobilization assays, MC148 had no effect on its own on any of the chemokine receptors, but in a dose-dependent manner blocked the stimulatory effect of the endogenous I-309 chemokine on CCR8 without affecting chemokine-induced signaling of any other receptor. In contrast, vMIP-II acted as an antagonist on 10 of the 16 chemokine receptors, covering all four classes: XCR, CCR, CXCR, and CX(3)CR. In chemotaxis assays, MC148 specifically blocked the I-309-induced response but, for example, not stromal cell-derived factor 1alpha, monocyte chemoattractant protein 1, or interleukin 8-induced chemotaxis. We thus concluded that the two viruses choose two different ways to block the chemokine system: HHV8 encodes the broad-spectrum chemokine antagonist vMIP-II, whereas MCV encodes a highly selective CCR8 antagonist, MC148, conceivably to interfere with monocyte invasion and dendritic cell function. Because of its pharmacological selectivity, the MC148 protein could be a useful tool in the delineation of the role played by CCR8 and its endogenous ligand, I-309.

Cloning, sequencing and variability analysis of the gap gene from Mycoplasma hominis.

The gap gene encodes the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The gene was cloned and sequenced from the Mycoplasma hominis type strain PG21(T). The intraspecies variability was investigated by inspection of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the gap gene from 15 strains and furthermore by sequencing of part of the gene in eight strains. The M. hominis gap gene was found to vary more than the Escherichia coli counterpart, but the variation at nucleotide level gave rise to only a few amino acid substitutions. To verify that the gene was expressed in M. hominis, a polyclonal antibody was produced and tested against whole cell protein from 15 strains. The enzyme was expressed in all strains investigated as a 36-kDa protein. All strains except type strain PG21(T) showed reaction to a 104-kDa band in addition to the expected 36-kDa band. The protein reacting at 104 kDa is a M. hominis protein with either an epitope similar to one on GAPDH, or it is an immunoglobulin binding protein.

Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity.

BACKGROUND: The association of Chlamydia pneumoniae with the development of atherosclerosis is based on serology and on detection of C pneumoniae-specific DNA by polymerase chain reaction in the atheromas. METHODS AND RESULTS: Because the humoral immune response frequently recognizes epitopes present on the surface of the bacteria, we analyzed what components are present on the C pneumoniae surface. We identified a family of proteins, the GGAI or Omp4-15 proteins, of which at least 3 are present on the surface of C pneumoniae. We immunized rabbits with recombinant GGAI proteins and used these antibodies in immunofluorescence microscopy of experimentally infected mice. In lung sections, a massive infiltration with polymorph nuclear neutrophil cells was observed. In the bronchial epithelial cells, C pneumoniae inclusions were seen. Evidence was found of differential expression of the GGAI proteins. CONCLUSIONS: On the basis of surface localization, differential expression, and the fact that the proteins are recognized by the human humoral immune response, we speculate whether these proteins, in addition to the lipopolysaccharides, are of importance for the immunopathogenesis of C pneumoniae.

The Mycoplasma hominis vaa gene displays a mosaic gene structure.

Mycoplasma hominis contains a variable adherence-associated (vaa) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolated by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N-terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequences boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.

Molecular biology of Mycoplasma.

Mycoplasmas are the smallest free living microorganisms with the smallest genome. The G+C content is in general low (25-33%) and the coding capacity is about 600 proteins. Mycoplasma species are phylogenetically related, they use the genetic codon UGA for tryptophan, and show rapid evolution, with a high rate of divergence. The genomes of Mycoplasma genitalium and Mycoplasma pneumoniae have been fully sequenced. Striking features of the M. genitalium sequencing project are the presence of a high number of membrane proteins with no resemblance to previously sequenced genes and the presence of repeated fragments of the gene encoding the tip-localized 140 kDa adhesin (MgPa). Many Mycoplasma species display a high frequency of antigenic variation, both as phase and size variation of individual antigens. Mycoplasma hominis isolates are known to be antigenic heterogeneous, as reflected in the reactivity with monoclonal antibodies (MAbs). The genetics of the antigenic variation has been studied for three different surface exposed antigens: P120, Lmp, and P50/Vaa. The gene encoding P120 had a hyper-variable region in the N-terminal region. In addition, a second gene with homology to p120 was identified. The gene encoding Lmp, a 135 kDa protein is repeated and both genes are translated and both contain internal repeated sequences. Deletion mutants in the lmp gene were obtained by cultivation of M. hominis PG21 with MAb 552 specific for the repeated part of Lmp. One of the lmp genes had deletions of from four to eight repeats. The other gene was left unaltered. The genes encoding P50/Vaa show a different form of variability where domains of the genes seem to be exchangeable. The genomic maps of five M. hominis strains showed that even though the size of the genomes varied the position of the different genes were in general conserved.

[Minor head injuries. Prognostic factors in the evaluation of patients]. / Lettere hovedtraumer. Prognostiske faktorer ved vurdering af patienter.

In order to identify signs to be used in the decision whether or not to admit patients after minor head injuries (MHI), the records of 713 females and 1163 males were reviewed. Skull X-ray was not obtained routinely, all patients were able to talk and walk when they reached medical contact. Nine patients developed an intracranial complication, three had an operation and one died. The risk of developing an ICC was 16.7% when the patient was agitated, 3.4% in the presence of impaired consciousness and 2.1% when positive neurological signs were observed at the time of examination. Based on the medical history, amnesia for more than five minutes and vomiting were associated with a risk of 3.3% and 1.2% respectively. The risk increased considerably in the presence of two of the above mentioned signs and was 60% if the patient was agitated and had amnesia for more than five minutes. Consequently, we recommend that all patients with one or more of the above symptoms or alcohol intoxication after a MHI should be admitted for observation. This policy may hold the possibility of considerable economic savings.

Prognostic signs in the evaluation of patients with minor head injury.

To assess signs that might be used in the decision whether or not to admit a patient with minor head injury, the records of 713 female and 1163 male patients were reviewed. Skull radiographs were not obtained routinely; all patients were able to walk and talk when they reached medical contact. Nine patients developed an intracranial complication. The risk of developing such a complication was 16.7 per cent when the patient was agitated, 3.4 per cent in the presence of impaired consciousness and 2.1 per cent when positive neurological signs were observed at the time of examination. Based on the medical history, amnesia for > 5 min and vomiting were associated with a risk of 3.3 and 1.2 per cent respectively; the risk increased considerably in the presence of both. It is recommended that all patients presenting themselves with one or more of the above symptoms or signs, or with alcohol intoxication, after a minor head injury be admitted for observation. If these guidelines had been used, all patients with an intracranial complication would have been detected, and 44.5 per cent of the bed-days used would have been saved.

Preoperative ultrasonographic verification of peritonsillar abscesses in patients with severe tonsillitis.

Infection around the tonsillar region does not always mean the presence of a peritonsillar abscess although the condition of peritonsillitis without abscess formation may clinically present similarly. It is, however, of therapeutic importance to distinguish between the two conditions. Treatment for abscess is surgical: aspiration, incision and drainage or immediate tonsillectomy. In contrast, phlegmonous peritonsillitis only requires antibiotics. In order to evaluate the diagnostic implications of preoperative ultrasonography in patients referred for treatment of peritonsillar abscess, 27 consecutive patients were subjected to bilateral ultrasound examination to visualize the tonsillar region. The transducer used was placed just below the mandibular angle, pointing posteriorly and cranially. The results of this study showed that it was possible to verify the presence of an abscess in approximately 90% of the cases. We suggest that this examination be performed whenever the normal clinical examination is insufficient due to trismus, lack of patient cooperation, etc.

[Minor head injuries in a Copenhagen district. 1. Epidemiology]. / Mindre hovedtraumer i et københavnsk omegnsområde. 1. Epidemiologi.

The records of 1,218 males and 743 females admitted to Copenhagen County Hospital in Glostrup between January 1 1985 and December 31 1986 were studied. The mean age was 28.2 years (SD = 20.3 years). The incidence rate was 360 per 100,000 inhabitants per year and was between 30 and 130% larger for males than females (p less than 0.001), the incidence rate was 26% higher in this study as compared to the rate in Denmark as a whole. Patients between 0 and 14 years were most frequently admitted between noon and the early evening hours and those between 15 and 64 years most frequently from late in the afternoon until the early night. The distribution was uniform throughout the week. There was a tendency towards more frequent admissions in the April and July trimesters. 74% of the patients were admitted at hours when the staff was minimal. This category comprised 4.9% of all patients admitted as emergencies to the hospital. The mean hospitalization was 1.6 days (range 1-14 days). Females were hospitalized longer than males (p less than 0.001). Furthermore, hospitalization was significantly longer, the older the patients were (p = 0.005). Patients with minor head trauma constitute a considerable work load in the hospital, especially outside normal working-hours.

[Minor head injuries in a Copenhagen district. 2. Causes, simultaneous lesions, alcohol and economic aspects]. / Mindre hovedtraumer i et københavnsk omegnsområde. 2. Arsager, konkurrerende laesioner, alkohol og økonomiske aspekter.

In order to elucidate causes, simultaneous lesions, alcohol intoxication and economic aspects of admission of patients with minor head traumas, the records of 1,961 patients admitted in 1985 and 1986 were reviewed. 35% were admitted after traffic accidents, 18% after assaults and 17% because of home accidents. In the age group 30-39 years, 40% of the males were admitted after assaults. 7% sustained fractures of the extremities, 4% facial fractures and 3% and 1% thoracic and abdominal lesions respectively. At least 28% of all patients, more than 50% of the patients admitted during evening- and night hours and 51% of the males between 14 and 65 years were clinically alcohol intoxicated. 1,876 patients were admitted solely for observation, nine developed an intracranial complication, three were operated upon and one died. The bed day expenditure was 2.7 mill. Dkr. (pounds 225,000), and for the entire nation 64.8 mill. Dkr (pounds 5,400,000). The costs for diagnosing one case of intracranial lesion in Denmark were 925.000 Dkr. (approximately pounds 80,000) and 2.1 mill. Dkr. (pounds 180,000) to find the cases requiring treatment (1985 level).

Is a skull X-ray necessary after milder head trauma?

During the 2-year period 1985-86 a total of 1,876 patients were admitted to our hospital after milder head trauma including cerebral concussion. Two hundred and eighty four patients who had a skull X-ray were not selected from guidelines. In 1,592 patients without a skull X-ray, signs of an intracranial complication developed in six cases and were verified by CT. In the 284 patients with skull X-ray a fracture was demonstrated in 25, and of these 25 patients only one patient disclosed a cerebral contusion. In the 259 patients with skull X-ray, but without demonstration of fracture, there were subsequently seen one subdural haematoma and one cerebral contusion. The incidence of intracranial complications in patients without and with skull X-ray with or without fracture does not differ significantly. In these circumstances we do not find any justification for routine skull X-ray after milder head trauma.