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Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin 0 (My) and 235 rRNA were sequenced for a range of Listeris monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multiloeus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.

Methods used to improve the accuracy of diagnosis of acute appendicitis are reviewed. Laparoscopy, barium enema, ultrasonography and computer assistance have all been shown to improve accuracy, but no one method is of proven superiority. Such diagnostic aids or intensive in-hospital observation must be used to reduce the 15-30 per cent negative laparotomy rate when acute appendicitis is suspected, without increasing the incidence of appendiceal perforation.

Polymerase chain reaction (PCR) primers for genus specific detection of Salmonella have been selected from a Salmonella-specific fragment of 2.3 kilobases (kb). Due to interserovar sequence diversity within this fragment, primer selection was based on DNA sequence alignment of sequences from 20 different Salmonella serovars. The specific PCR product of 429 base pairs (bp) was formed from 144 of 146 salmonella strains tested (116 of 118 serovars). The two false-negative strains belonged to two different serovars of the rarely isolated subspecies IIIa (monophasic S. arizonae). No product was produced in any of 86 non-Salmonella Enterobacteriacea strains tested, covering 41 species from 21 genera.

The gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5' flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3' end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).

The gene, flaA, encoding the flagellin protein of Listeria monocytogenes (strain 12067) has been isolated from an expression library in Escherichia coli using a flagellin-specific monoclonal antibody. DNA sequence analysis of a positive clone revealed the presence of an open reading frame of 287 amino acid residues with a calculated molecular mass of 30.4 kDa. Comparison of this sequence with flagellins from other bacteria showed a significant degree of homology in both the N- and C-terminal parts of the protein. The flagellin mRNA was determined to be 1 kb in size, which is the expected size for a monocistronic mRNA, and the temperature-dependent expression of flagellin was found to be regulated at the transcriptional level. Southern blot analysis, using the flagellin gene as probe, indicated that L. monocytogenes can be divided into two groups. These groups correspond to the flagellar antigens AB and ABC, respectively, as well as to the two types of L. monocytogenes based on the DNA sequence of the listeriolysin gene.

A new method for the extraction of bacterial DNA from soil has been developed. Soil samples of 50 g were dispersed, and bacteria were released by use of a cation-exchange resin; subsequently, bacteria were separated from soil particles by low-speed centrifugation and lysed with lysozyme and ionic detergent, and the DNA was then purified by CsCl-ethidium bromide equilibrium density centrifugation. The extracted DNA was of high molecular weight and sufficiently pure for restriction enzyme digestion, DNA-DNA hybridization, and amplification by the polymerase chain reaction. The advantages of the new method are that the separation of bacteria from soil is considerably faster than by repeated blending, more samples can be handled, and furthermore no aerosols are formed during separation. Also, we investigated whether the CsCI-ethidium bromide equilibrium density centrifugation could be replaced by purification using Gene-Clean. However, this method produced DNAs which were insufficiently pure for several types of analysis. The new method was used to study survival of a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading Pseudomonas cepacia DBO1 (pRO101) in unamended soil and in soil amended with 2,4-D. We found that the degrading strain, irrespective of inoculation level, was able to grow to the same high numbers in soil amended with 2,4-D, while the strain in nonamended soil were maintained at the inoculation level. Detection based on DNA extraction and subsequent dot blot DNA-DNA hybridization was in accordance with detection by plating on selective medium.

ObjectiveTo evaluate the long-term results of implantation of an artificial anal sphincter (AAS) for severe anal incontinence. Summary Background DataImplantation of an AAS is one of the options for treatment of anal incontinence when standard operations have failed. It is the only surgical option for treatment of anal incontinence in patients with neurologic disease that affects the pelvic floor and the muscles of the lower limb. MethodsSeventeen patients underwent implantation of an AAS before 1993. These patients have been followed and their continence status evaluated. ResultsTwo patients died of unrelated causes within the first 3 years after surgery, and in three patients the AAS was explanted because of infection. During the follow-up period, four patients had the AAS removed because of malfunction, and eight patients had a functioning AAS Ն5 years after the primary implantation. Five of these patients had revisional procedures, mainly because of technical problems in the early part of the study, when a urinary sphincter or slightly modified urinary sphincter was used. Continence at follow-up was good in four patients and acceptable in three, whereas one patient still had occasional leakage of solid stool. One patient had rectal emptying problems, which she managed by enema. ConclusionsAn AAS based on the same principles as the artificial urinary sphincter seems to be a valid alternative in selected patients when standard surgical procedures have failed or are unsuitable. Approximately half of the patients have an adequate long-term result. Infectious complications still present a problem, whereas mechanical problems are less frequent with the modification of the device now available.Treatment of severe anal incontinence by implantation of an artificial anal sphincter (AAS) was first reported by us in 1987, 1 and the first series were published in 1989 and 1992.2,3 The first patients were implanted with an unmodified urinary sphincter, but subsequently this device was modified to meet the demands of a bowel sphincter (higher closing pressure and increased strength of the cuff).Because production of the sphincter was suspended, only a few patients underwent implantation until 1996, when the device again became available to a few centers. Recently published results from these centers have confirmed our early positive evaluation of the method. 4 -6 The follow-up varied from 4 to 76 months in the first two of these series 4,5 and from 4 to 12 months in the last. We herein present the first long-term results with a follow-up of Ն5 years with an AAS. PATIENTS AND METHODSBetween 1987 and 1993, 17 patients (11 women and 6 men; median age 46 [range 32 to 65 years]) underwent implantation of the AAS. The first six patients received a urinary sphincter (AMS 800, American Medical Systems, Minneapolis, MN); the last 11 received a modified version of the urinary sphincter (the cuff-tab was strengthened, the cuffs were made wider, and the pressure-regulating balloon was enlarged so that a cuff pressure of up to 90 cm H...