Biomedical subjects
Publications and source records attributed to J R Scott.
Accurate assessment of flexor tendon function in a digit with an acute, non-penetrating injury is difficult. MR imaging can negate the need for surgical exploration and the associated morbidity.
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The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.
PROBLEM: The results regarding human leukocyte antigen (HLA) DQ alpha allele sharing in recurrent miscarriage couples are conflicting. The purpose of this study was to determine the frequency of HLA DQ alpha allele sharing in our unexplained recurrent spontaneous abortion (RSA) patients using modern DNA analytical techniques. METHODS: DNA was extracted from whole blood samples of 1) 51 couples with at least three miscarriages, and 2) 43 fertile couples (with at least seven children and no known history of recurrent miscarriage). The polymerase chain reaction (PCR) was used to amplify the second exon of the HLA DQ alpha locus on chromosome 6. Genotypes were identified by allele specific hybridization with 12 sequence-specific oligonucleotide probes. RESULTS: 47% of recurrent miscarriage couples and 35% of fertile couples shared no alleles. 47% of recurrent miscarriage couples compared to 58% of fertile couples shared one allele, and 6% of recurrent miscarriage couples and 7% of fertile couples shared two alleles. CONCLUSIONS: Reproductive partners with unexplained recurrent pregnancy loss have no increased frequency of HLA DQ alpha allele sharing. It is unlikely that HLA DQ alpha genotyping will be helpful in the management of patients with RSA.
Many Streptococcus pyogenes immunoglobulin-binding proteins have structural similarities to the antiphagocytic M protein, including the well-known C repeats. One of these molecules is the immunoglobulin A-binding protein Arp, which is expressed by a serotype 4 strain for which no antiphagocytic M protein has yet been described. We expressed Arp4 in an S. pyogenes strain from which the structural gene for the M protein has been deleted and found that Arp4 is not sufficient to inhibit phagocytosis.
Transcription of the gene encoding the antiphagocytic M protein (emm) of the group A streptococci has been shown to be regulated by CO2 (M. G. Caparon, R. T. Geist, J. Perez-Casal, and J. R. Scott, J. Bacteriol. 174:5693-5701, 1992). We tested the influence of additional environmental growth conditions on emm expression. Increased osmolarity, low temperature, growth with free exchange of gasses, or the restricted availability of iron resulted in decreased transcription from the emm promoter.
We have cloned and sequenced the DNA needed for production of CS2 pili in Escherichia coli K-12. The four open reading frames, cotB, cotA, cotC, and cotD, show homology with the genes needed for production of CS1 and CFA/I pili, which are also found on enterotoxigenic E. coli associated with human diarrheal disease. We also report that CotA plus CotB interact with the CS1 gene products CooC and CooD to form pili that can be visualized by electron microscopy and, conversely, that the CS1 gene products CooA and CooB interact with CotC and CotD to form pili.
Transcription of the surface-associated virulence factors of the group A streptococcus (GAS) Streptococcus pyogenes, M protein (emm) and the C5a peptidase (scpA), is activated by a protein called Mga (formerly Mry or VirR). To determine whether Mga binds directly to the promoters of the genes it regulates, a protein resulting from the fusion of Mga to the C-terminal end of maltose-binding protein was purified from Escherichia coli. Specific binding to the promoter regions of the scpA and emm alleles of the type M6 GAS strain JRS4 was demonstrated by electrophoresis of the DNA-protein complex. Competition studies showed that the region upstream of scpA bound MBP-Mga with a slightly higher affinity than did the region upstream of emm. DNase I protection experiments identified a single 45-bp binding site immediately upstream of and overlapping the -35 region of both promoters. Sequences homologous to the protected regions were found in the promoters of many emm, scp, and emm-like genes from strains of different serotypes of GAS, and a consensus Mga binding site was deduced.
Conjugative transposons are important determinants of antibiotic resistance, especially in gram-positive bacteria. They are remarkably promiscuous and can conjugate between bacteria belonging to different species and genera. Transposon-promoted conjugation may be similar to F plasmid-promoted conjugation, as it appears that only one strand of the transposon DNA is transferred from donor to recipient. The recent determination of the entire nucleotide sequence of Tn916 allowed us to make specific predictions about the possible function of different open reading frames and the position of a (hypothetical) origin of transfer. The mechanism of recombination during conjugative transposition differs from that of other transposons, as shown by the absence of a duplication of the target sequence upon integration. The current model for recombination postulates that staggered double-stranded cleavages occur at each end of the transposon. One DNA strand is cut six bases from the end of the transposon, and the other strand is cut immediately adjacent to the end. The ends of the excised transposon are then ligated to form a circular intermediate with a six-base heteroduplex. Staggered cleavages of the circular intermediate and the target DNA allow the transposon to insert into the target, where it is flanked by heteroduplex regions that are resolved by replication. All hosts examined contain preferential target sites: these are not specific sequences but apparently consist of bent DNA. The site-specific recombinases encoded by conjugative transposons belong to the integrase family. Like phage lambda integrase, the integrase of Tn916 has two DNA-binding domains that recognize different sequences, one within the ends of the element and one that includes target DNA. The affinity of Tn916 integrase for target sites correlates with the frequency of integration into a particular site. The similarity between conjugative transposons and phage lambda is striking and suggests that both use the same mechanism of recombination. In lambda, however, recombining sites must be homologous. Homology may be necessary because of branch migration, which is thought to occur during recombination. In conjugative transposition, the recombining sites are nearly always different, and therefore branch migration probably does not occur. This review presents a speculative model for the alignment of the ends of Tn916 during excision that was adapted from one recently proposed for lambda.
We studied 108 adult cases of elective lumbar surgery using dermatomal somatosensory-evoked potential (DSEP) monitoring to evaluate its usefulness due to concern over potential neurologic injury during pedicle screw insertion. Both surgeons used all of the necessary precautions required during surgery so that DSEP monitoring was not the "primary," but rather a backup system for operative security. Quality tracings were obtained in 71% of cases; anesthetic difficulties being the major cause of poor monitoring. There were no neurological complications related to pedicle screw insertion. We found that DSEP monitoring was an excellent method to verify intraoperative neurological status, but required a high degree of cooperation between the anesthesiologists, monitoring technician, and surgeons. In today's cost-containment environment, its usefulness is subjected to the expertise of the spine surgeon and the hospital setting.
The transmissible neurodegenerative diseases, of which scrapie is the archetype, are caused by unconventional infectious agents. Prion protein (PrP), a widespread host coded, cell surface sialoglycoprotein, is thought to be an essential or, controversially, sole component of these agents. During infection, disease specific accumulations of PrP may be observed in immunostained brain sections of mice infected with the 87V scrapie strain as amyloid plaques or as diffuse or granular foci within the neuropil. Using serial light and electron microscopical preparations we determined immunocytochemically that infection specific PrP is present in amyloid fibrils, and accumulates on the plasmalemma of neurites at the periphery of plaques and in the neuropil, irrespective of the morphological form of PrP accumulation when viewed by light microscopy. In some brain areas with dense granular PrP expression complete disruption of neuropil with loss of neurites was associated with fibrils lying free in expanded extracellular space. These results suggest that normal PrP may be converted to its pathological form at the neuronal plasmalemma or in the extracellular space and, furthermore, that amyloid fibrils are formed following the accumulation and aggregation of subunit proteins at these sites.
An originally heretical proposition that the transmissible spongiform encephalopathies are caused by a host-coded protein (the prion hypothesis) is now current dogma. Indeed these disorders are commonly called prion diseases but the prion hypothesis provides no readily acceptable explanation for the source of the informational component of the agent necessary to code for the diversity of strains of scrapie. Ultrastructural immunolocalisation of prion protein (PrP) in murine scrapie shows that PrP accumulates in association with the plasmalemma of neurones, diffusing from the neuronal cell surface into the extracellular space around small neurites prior to aggregation and fibril assembly. These events occur without the involvement of other cell types. The area of neuropil infiltrated with extracellular PrP around infected neurons and neurites indicates that the form of PrP initially produced is not immediately amyloidogenic.
Prion protein (PrP) is an abundant membrane-associated host protein which accumulates in abnormal, relatively protease-resistant forms in the brains of animals with scrapie and related diseases. Using correlative light and electron microscopy we determined the sites of subcellular localization of PrP in mice infected with the 87V strain of scrapie. Disease-specific accumulation of PrP was observed at light microscopy as amyloid plaques or as diffuse or granular staining within the neuropil, often clearly associated with individual neurons. Serial electron microscopical preparations were immunostained for PrP by the immunogold method. Gold particles were located on amyloid fibrils and on the plasmalemma of neurites at the periphery of plaques and in the neuropil, irrespective of the morphological form of PrP accumulation when viewed by light microscopy. This suggests the amyloid fibrils are formed following the accumulation and aggregation of sub-unit proteins at the plasmalemma and, furthermore, that normal PrP may be converted to its pathological form at this site.
This study was undertaken to determine to what extent neuronal loss is a feature of scrapie pathology, using an experimental model in which infectivity and subsequent vacuolar lesions are well characterized but in which neuronal loss has not been previously identified. Intraocular infection with ME7 scrapie directs infection through the major projections of the optic nerve, which include the dorsal lateral geniculate nucleus (dLGN) on the contralateral side to the infected eye. Infectivity can be detected in the dLGN at 77 days post-infection and vacuolar lesions are first seen around halfway through the incubation period of 240 days. Morphometric assessment of neuron number in the dLGN was made on gallocyanin stained semi-serial sections from 5 infected and 5 normal brain-injected controls at 4 fifty-day intervals during the incubation period, and on clinically terminal mice. The number of neurons in the dLGN of the infected mice decreased steadily from around 20,000 at 50 days post-infection to under 2,000 in the terminal group. The loss was delayed in the ipsilateral dLGN, although terminal counts were the same for both sides. The onset of neuronal loss was coincident with initial vacuolar changes, and neuronal numbers were inversely proportional to the severity of vacuolation. It is concluded that scrapie infection causes a progressive neuronal loss that can be identified some 30-80 days after infectivity can be detected in the dLGN, long before the onset of clinical disease.
It is difficult to determine whether alloimmune factors are responsible for some cases of recurrent miscarriage. No diagnostic tests have been found to be clinically useful. It appears that paternal cell immunization may increase the live birth rate slightly, but the results of large prospective, randomized studies presently being conducted are necessary to confirm this therapeutic effect. Finally, there are few published reports on the neonatal, childhood, or long-term effects of immunotherapy on the offspring. Therefore, we recommend that physicians adopt a cautious approach to the diagnosis and treatment of potential alloimmune RPL. Patients who strongly desire to pursue immunotherapy after appropriate counseling should be referred to a research center with an interest in this area.
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Transposition of conjugative transposons proceeds by excision and formation of a covalently closed circular intermediate that includes at its joint the six flanking bases from its previous host (coupling sequences). To elucidate the role of the coupling sequences in this process and to determine the sequence of targets used by Tn916, we studied its insertion into a plasmid following conjugation. The results differ from those previously observed when Tn916 was introduced by transformation. They suggest that only one specific strand of the transposon molecule is transferred during the conjugation event and that complementary strand synthesis produces a double-stranded transposon circle with no mismatches which serves as the reaction intermediate. Tn916 inserts preferentially at specific sites and the same targets are used when Tn916 comes from donors with different coupling sequences. An analysis of the sequences of preferred targets is presented.
Many strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces. These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple identical protein subunits encoded by cooA. We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA. Following cooD is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E. coli K-12 to synthesize CS1 pili. Complementation analysis of mutants cloned in E. coli K-12 and constructed in an ETEC-derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili.