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Objective: This study aimed to establish a practical diagnostic test for choroideremia (CHM) and to show its application in a family with the clinical diagnosis of choroideremia., Design: Case series., Participants: Sixteen males from 13 families with clinically documented CHM and unaffected normal males were enrolled in this study., Methods: Protein extracted from either leukocytes or Epstein-Barr virus-transformed lymphocytes was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of the protein was performed with two monoclonal antibodies, one against the CHM gene product, Rab escort protein-1 (REP-1), and the other against the alpha-subunit of farnesyl transferase. DNA was extracted from peripheral leukocytes and subjected to polymerase chain reaction-single stranded conformation polymorphism analysis using primers for the exons of the CHM gene. Where altered mobility of the DNA fragments was detected, direct sequencing of that exon was compared with the published normal sequence., Results: The authors detected REP-1 in protein samples extracted from lymphoblastoid cell lines from female carriers but not from CHM males. The authors also showed the absence of REP-1 in the peripheral leukocytes of males affected with CHM. In one male who lacked REP-1, direct sequencing of exon 7 showed a cytosine-to-thymine transition mutation (Arg293X) in the CHM gene., Conclusions: The clinical diagnosis of CHM can be confirmed simply by immunoblot analysis with anti-REP-1 antibody, showing the absence of REP-1 protein in peripheral blood samples. Because all known mutations in the CHM gene create stop codons that truncate the protein product and result in absence of REP-1, the authors predict that most patients with CHM can be diagnosed by this procedure.

Popular misconceptions view economic crime against workers' compensation systems as victimless and therefore acceptable. This chapter argues that the courts must express strong denunciation of this fraud to engender a climate of deterrence. The law and order approach developed in Alberta, where Workers' Compensation Board investigators are given police powers, is examined.

Recently, the clinical application of electronic portal imaging devices has enabled more frequent verification of patient setup for radiation treatment. However, the image quality has sometimes proven to be inadequate, motivating the investigation of alternative sensors with better image quality. Amorphous selenium (a-Se) is potentially one such sensor since the electrostatic image formation process has high resolution. To fully evaluate the potential of a-Se for portal imaging, it is necessary to investigate all the imaging properties at high x-ray energies. Here, measurements of the sensitivity of a-Se to incident x-ray spectra ranging in energy from 40 kVp to 18 MV and for a-Se thicknesses ranging from approximately 10 to 300 microns under full buildup conditions are described. When x rays or energetic electrons deposit energy in a photoconductor with an applied electric field, F, electrons and holes are released. The x-ray conversion sensitivity may be defined as 1/W +/-, where W +/- is the energy required to release an electron-hole pair. Consistent with the results of previous investigators, W +/- is found to vary approximately with F-2/3. Unexpectedly, over the energy range of 40 kVp to 18 MV, W +/- was found to decrease by a factor of nearly 3. These dependencies are compared to the predictions of two competing charge recombination models, geminate and columnar. The results are explained by a microdosimetric model in which the sensitivity at megavoltage energies is governed by geminate recombination, but at lower energies, both mechanisms are involved. Thus, the sensitivity of a-Se to x rays spanning the diagnostic and radiotherapy range has been measured and the physical basis for this behavior established.

We have generated a panel of deletion mutants of ors12 (812-bp), a mammalian origin of DNA replication previously isolated by nascent strand extrusion from early replicating African Green monkey (CV-1) DNA. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the Dpnl resistance assay, using extracts from HeLa cells. We identified a 215-bp internal fragment as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and CTTA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats. 22 and 16 bp long, respectively. The overall sequence of the 215-bp fragment is G/C-rich (50.2%), by comparison to the 186-bp (33.5% G/C-rich) minimal sequence required for the autonomous replication activity of ors8, another functional ors that was similarly isolated and characterized.

Complex nonadditive interactions between specific alleles at multiple loci may underlie many so-called multifactorial threshold birth defects. The open-eyelids-at-birth defect in mice is a good model for these defects, and an understanding of its genetic complexity begins with mapping the participating loci. The open-eyelids defect can be part of a syndrome or can occur with no other obvious phenotypic effects. Of the latter nonsyndromic forms, the lidgap series includes four extant mutations that are considered to be alleles based on complementation tests. All show genetic complexity in segregation ratios. None has been mapped previously. On the basis of a strategy of mapping the mutation with the simplest inheritance pattern first, we generated an extensive exclusion map for lidgap-Gates, lgGa, using morphological and protein polymorphisms. We then screened the non-excluded regions in a congenic strain, AEJ.LGG-lgGa, for SSLP markers and located the differential chromosome segment containing the lgGa locus in a region near the distal end of mouse Chromosome (Chr) 13. This linkage was confirmed and refined by typing SSLPs in 64 F2 and 74 BC1 progeny of a cross of LGG/Bc (lgGa/lgGa) to SWV/Bc. The lgGa mutation maps to a 1- to 2-cM region between D13Mit76 and D13Mit53. Integrin alpha 1 and integrin alpha 2, which map to the same general region, are possible candidate loci, based on their embryonic expression and cellular function. Evidence is also presented for a common unlinked recessive suppressor of the open eyelids trait caused by lgGa.

In fluoroscopic portal imaging systems, a metal plate is bonded to a phosphor screen and together these act as the primary x-ray sensor. The light from the screen is collected and imaged by a lens on the target of a video camera. The demagnification (M) between the large area of the phosphor being imaged and the small active area of the video camera results in poor optical coupling between the screen and the video camera. Consequently x-ray quantum noise is small compared to other noise sources. By reducing the demagnification, the light from the screen is collected more efficiently, so we were able to increase the x-ray quantum noise relative to other noise sources and thus unambiguously identify it. The noise power spectrum was measured as a function of M to determine the relationship between the x-ray quantum noise. shot noise, and amplifier noise. It was found by extrapolation to clinical demagnifications that the amplifier noise dominates x-ray quantum noise, at all spatial frequencies, but the shot noise was less than the x-ray quantum noise at low spatial frequencies. For low spatial frequencies, this implies that a secondary quantum sink can be avoided. If amplifier noise could be sufficiently reduced, x-ray quantum limited images could be obtained in clinical systems at low spatial frequencies.

Cleft lip with or without cleft palate, CL(P), a common human birth defect, has a genetically complex etiology. An animal model with a similarly complex genetic basis is established in the A/WySn mouse strain, in which 20% of newborns have CL(P). Using a newly created congenic strain, AEJ.A, and SSLP markers, we have mapped a major CL(P)-causing gene derived from the A/WySn strain. This locus, here named clf1 (cleft lip) maps to Chromosome (Chr) 11 to a region having linkage homology with human 17q21-24, supporting reports of association of human CL(P) with the retinoic acid receptor alpha (RARA) locus.

The pathology of Mycoplasma pneumoniae pulmonary infection for a hamster model was examined after whole bacterium rechallenge or component vaccination. Animals which, after an initial infection, were rechallenged with either live or heat-killed M. pneumoniae inocula developed severe early recall lesions in the first 3 days. In contrast, animals infected once develop maximum histopathology at approximately 10-14 days. A severe perivascular inflammatory cellular infiltrate developed in the rechallenged groups, and pulmonary pathology could also be elicited by rechallenge with bacterial growth medium components. Component vaccination with protein P1 did not reduce disease in comparison to once-infected controls, and vaccination promoted an early immune recall response as well. We conclude that an early immune response needs to be sought in all future experiments of challenge/rechallenge or vaccination. Vaccine studies will require an understanding of both protective and harmful immunogens.

Univariate and multivariate analyses were applied to determine risk factors for the progression of Escherichia coli O157:H7 enteritis to hemolytic uremic syndrome (HUS). Both clinical and laboratory variables were assessed for 118 pediatric patients (28 HUS; 90 enteritis only). Verotoxins 1 and 2 were produced by 89% of E. coli strains whereas verotoxin 2 only was produced by 11%. Although a greater frequency of strains producing verotoxin 2 only occurred in HUS isolates (p = 0.11), toxin phenotype was not significantly associated with risk after multivariate analyses. HUS patients with or without neurological manifestations had similar frequencies of the two toxin phenotypes among their isolates. Significant associations for young age (RR = 0.984; 95% CI = 0.971-0.998) and prolonged use of antidiarrheal agents (RR = 44.11; 95% CI = 8.48-229.4) with HUS were apparent. A lesser chance of progression was observed for patients whose strains possessed a 4 kb plasmid (RR = 0.27; 95% CI = 0.08-0.94). Our results are consistent with the hypothesis that progression to HUS is dependent upon both bacterial virulence factors and the clinical characteristics of the individual patient.

Serum samples from 49 children with acute Mycoplasma pneumoniae infection were screened for the presence of antibodies to mitotic spindle apparatus. None of these serum samples showed such antibodies at a screening dilution of 1:40, though anticentriolar antibodies at titres of 1:320 were observed in two children with acute cerebellar dysfunction. Anticentriolar antibodies may play a part in the pathogenesis of CNS disease associated with M pneumoniae infection.

Three immunodominant antigens of Streptococcus equisimilis (Lancefield group C) with approximate mol. wts of 46, 66 and 105 kDa were recognised by human serum IgG and IgA immunoblotting. These antigens were identified consistently by various human sera but immunoblots with IgA (heavy chain) and secretory IgA (J chain) from human respiratory secretions gave more variable results. Antigens with similar migration rates were demonstrated in S.pyogenes, large colony human biotype group G streptococci, and streptococci of groups C and G from the "S. anginosus-milleri group". Polyclonal antibody which was eluted from immunoblot substrates that contained the S. equisimilis 66-kDa antigen reacted with the 60-kDa antigen of S. pyogenes. Both polyclonal and monoclonal anti-vimentin antibodies identified the 46-kDa and 66-kDa antigens of S. equisimilis. The homology of these antigens among beta-haemolytic streptococci has the potential to complicate both a strategy for the utilisation of immunoblotting for diagnostic purposes and the understanding of how such antigens may be involved in the pathogenesis of post-infectious sequelae.

Flagellin proteins from several different strains of Pseudomonas pseudomallei have been isolated and purified to homogeneity by mechanical shearing and differential centrifugation techniques. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded flagellin monomer protein bands with an estimated M(r) of 43,400. No lipopolysaccharide contamination of the purified protein preparations was detectable by silver staining of flagellin displayed on polyacrylamide gels and by Western immunoblotting with P. pseudomallei antilipopolysaccharide monoclonal antibody. NH2-terminal amino acid sequence analysis of the flagellin protein of P. pseudomallei 319a revealed significant homology with flagellins from Proteus mirabilis, Bordetella bronchiseptica, and Pseudomonas aeruginosa PAK. Rabbit polyclonal antiserum raised against the 319a flagellin protein reacted with 64 of 65 P. pseudomallei strains tested. The polyclonal antiserum proved effective in inhibiting the motility of these organisms in motility agar plates. Passive immunization studies demonstrated that 319a flagellin-specific antiserum was capable of protecting diabetic rats from challenge with a heterologous P. pseudomallei strain.

Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.

The contrast and zero spatial frequency signal-to-noise ratio produced by a method for radiation therapy portal imaging known as low-energy imaging with high-energy bremsstrahlung beams have been mathematically analyzed. The analysis makes extensive use of Monte Carlo techniques and incorporates the detector, the spectrum, phantom, and geometry. The analysis is validated through comparison with measured data including subject contrast measurements and the attenuation of the beam with lead. Scatter reduction is found to be potentially the most effective method to improve contrast and SNR for a film based system. A large fraction of the scatter detected is of a much higher energy than that found in diagnostic radiology. Hence, traditional antiscatter grids, such as those used in diagnostic radiology, are ineffective. The analysis and theory from the literature are applied to design a new grid which is more appropriate for this application. The grid produces a modest improvement according to a contrast-detail study.

ors12, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. Zannis-Hadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721-727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. Natl. Acad. Sci. USA 84, 6668-6672, 1987) and in a cell-free system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of ors12 (nucleotides 1-168) consists of the highly reiterated alpha-satellite sequence (B. S. Rao et al. Gene 87, 233-242, 1990). We have estimated the copy number of the non-alpha-satellite portion of ors12 in CV-1 cells to be < 9 copies per haploid genome and have used it as a probe to generate a genomic map of ors12 on CV-1 DNA. In situ hybridization of CV-1 metaphase chromosomes, using a biotinylated probe of the entire ors12 sequence, positively identified the centromeres of all chromosomes. However, when the non-alpha-satellite portion of ors12 was used as a probe, it positively identified the centromeric region of only six chromosomes, namely, B4, C11, D14, D24, E25, and E27, as well as that of a marker chromosome. The results suggest that ors12 represents a centromeric putative replication origin that is present on a subset of CV-1 chromosomes and is activated at the onset of S phase.

The gene for Huntington disease (HD) has been localized close to the telomere on the short arm of chromosome 4. However, refined mapping using recombinant HD chromosomes has resulted in conflicting findings and mutually exclusive candidate regions. Previously reported significant nonrandom allelic association between D4S95 and HD provided support for a more proximal location for the defective gene. In this paper, we have analyzed 17 markers, spanning approximately 6 Mb of DNA distal to locus D4S62, for nonrandom association to HD. We confirm the previous findings of nonrandom allelic association between D4S95 and HD. In addition, we provide new data showing significant nonrandom association between HD and 3 markers at D4S133 and D4S228, which are approximately 3 Mb telomeric to D4S95.

To identify expressed sequences within candidate regions for the Huntington's disease (HD) gene in 4p16.3, we isolated the gene encoding the beta subunit of the human cGMP phosphodiesterase (PDEB). We formally assessed this as a candidate gene for HD based on it's expression in brain, the demonstration of linkage disequilibrium between intragenic DNA markers and HD, and the demonstration that mice with a mutation in this gene have a reduction of neurons in particular brain regions. We investigated all 22 exons of PDEB and 5'-flanking region for point mutations in 16 HD patients of different ethnic origins using single strand conformational polymorphism analysis. The underlying DNA changes found initially exclusively in HD patients were excluded as the cause for HD.

A histopathological scoring system was developed to assess the pathology of acute Mycoplasma pneumoniae pulmonary infection in a hamster model. A final score per animal (ranging 0-26) is obtained by averaging scores from each lung which have been accumulated by the addition of subscores from the assessments of quantity and quality of peribronchiolar and peribronchial infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. The scoring scheme was then applied to test the ability of a heat-killed inoculum to induce pulmonary pathology and to the trial of a 43 kDa protein-associated antigen as a vaccine immunogen. A heat-killed inoculum delivered by both intratracheal and intranasal routes did not induce pulmonary pathology compared to a live inoculum (respective mean scores 0.1, 6.7; P less than 0.01). Animals prevaccinated with the 43 kDa antigen developed an accentuated pathological response after live challenge compared to those unvaccinated (respective mean scores 16.8, 5.8; P = 0.00007). Hypersensitization to growth medium components may, however, have contributed to the accentuated disease since the lungs of vaccinated animals challenged with culture-negative media also were affected (mean score 5.4). Reproducibility of the scoring system was measured by duplicate reading of histology slides which were randomized to the observer upon the second reading (r = 0.93; P = 0.000009). The scoring system has the ability to differentiate disease severity in small groups of animals.

Methylumbelliferyl-conjugated enzyme substrates were assessed for their ability to differentiate beta haemolytic streptococci in Lancefield groups C and G. Both Streptococcus equisimilis (group C) and large colony human biotype group G strains were consistently differentiated from group C and G "Streptococcus milleri group" bacteria by their ability to hydrolyse the beta-D-glucuronide substrate. The test was completed in less than one hour.

The oligomerization domain of the reovirus cell attachment protein (sigma 1) was probed using the type 3 reovirus sigma 1 synthesized in vitro. Trypsin cleaved the sigma 1 protein (49K molecular weight) approximately in the middle and yielded a 26K N-terminal fragment and a 23K C-terminal fragment. Under conditions which allowed for the identification of intact sigma 1 in the oligomeric form (approximately 200K) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the N-terminal 26K fragment was found to exist as stable trimers (80K) and, to a less extent, as dimers (54K), whereas the C-terminal fragment remained in the monomeric form. A polypeptide (161 amino acids) containing the N-terminal heptad repeat region synthesized in vitro was capable of forming stable dimers and trimers. Using various criteria, we demonstrated that the stability of the intact sigma 1 oligomer is conferred mainly by the N-terminal heptad repeat region. Our results are summarized in a model in which individual heptad repeats are held together in a three-stranded alpha-helical coiled-coil structure via both hydrophobic and electrostatic interactions.

The N-terminal portion of the reovirus cell attachment protein sigma 1 has recently been shown to possess intrinsic oligomerization and virion-anchoring functions. Sequence analysis of the sigma 1 proteins of the three reovirus serotypes has revealed the presence of distinct structural domains within this region: a terminal hydrophobic tail, a hinge, and an extended coiled-coil. To probe the inter-relationship between the virion-anchoring function and the oligomerization function, we constructed two serotype 3 (T3) sigma 1 deletion mutants in SV40 expression vectors, one lacking the hydrophobic tail and the hinge, and the other lacking an adjacent region which constituted part of the coiled-coil. These mutants were (i) expressed in uninfected COS-1 cells and assayed for their ability to form oligomers, and (ii) expressed in type 1 (T1) reovirus-infected COS-1 cells and assayed for their incorporability into progeny T1 virions. It was found that, whereas both truncated sigma 1 proteins were capable of forming stable oligomers, neither could be incorporated into virions. These observations, coupled with structural characteristics deduced from sequence analysis, are compatible with a model in which the hydrophobic tail is the bona fide sigma 1 anchorage domain and whose precise association with the virion spikes is dictated by an adjacent heptad repeat region linked to the former structure via a flexible hinge region.

Previously the receptor recognition domain of the reovirus serotype 3 (T3) cell attachment protein (sigma 1) was mapped to the C-terminal half of the protein using deletion mutagenesis of the reovirus S1 gene. A similar approach has been adopted in the present study to map the domain on T3 sigma 1 that is responsible for incorporation into the virion (i.e., the anchoring domain). Restriction enzymes which divide the T3 S1 cDNA into four segments (5'-I-II-III-IV-3') of similar size were used to generate four mutants, each with a particular segment deleted. The mutants were cloned into SV40 expression vectors and used to transfect COS-1 cells which were subsequently with reovirus serotype 1. Progeny viral particles with truncated T3 sigma 1 proteins incorporated were then identified by radioimmunoprecipitation with a serotype-specific anti-T3 sigma 1 serum. It was found that the mutant lacking I (mutant dl) was totally incapable of being incorporated into the virion, whereas the mutant lacking domain II (mutant dII) was incorporated efficiently. Due to altered antigenicities of the mutants lacking domain III (mutant dIII) or domain IV (mutant dIV), incorporation of these two proteins into virions was less detectable using the above assay. Evidence that domain I (the N-terminal 121 amino acids) alone dictates the incorporation of sigma 1 into the virion came from the subsequent demonstration that a chimeric protein containing domain I fused to chloramphenicol acetyltransferase (CAT) was incorporated into the virion (detectable with an anti-CAT serum) as efficiently as the full-length sigma 1 protein.

A rapid immunoblotting technique based on the IgM response to a major immunogenic protein is described for the diagnosis of Mycoplasma pneumoniae infection. Using results of the complement fixation test as the criterion for diagnosis, the rapid immunoblot method was positive in 95.7% of patients. The sensitivity was reduced to 81.9% if the test was performed on either single sera or acute sera only from serum pairs. Although the few sera that failed to demonstrate a positive IgM response were more likely to be from older patients, there was a consistent IgM response recorded for both younger (less than 20 years) and older (greater than or equal to 20 years) patients.

The complete sequence of the reovirus (serotype 3) S1 gene was obtained by using cloned cDNA derived from the RNA segment. This gene is 1416 nucleotides in length and contains two open reading frames. The first reading frame has a coding capacity of 455 amino acids, sufficient to account for the known S1 product, protein sigma 1 (42,000 MW). It possesses a signal peptide as well as three possible glycosylation sites. No homology could be detected when this gene sequence and the deduced amino acid sequence were compared to published sequences of the corresponding gene of a human rotavirus. The second reading frame (not in phase with the first) starts at the second ATG recently shown to be a functional initiation site. It has a coding capacity of 120 amino acids. Its outstanding feature is the highly basic amino-terminal region, a characteristic apparently shared by a number of DNA binding proteins. It is speculated that this protein, hitherto undetected, may play a role in mediating viral and/or host nucleic acid transcription.

A cDNA clone encompassing the complete reovirus (serotype 3) S1 gene was constructed using two partial clones containing overlapping sequences. The gene (with the first 15 bases at the 5' end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter. The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-sigma 1 antibodies. Like authentic soluble sigma 1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors. Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E. coli cells, were also found to exhibit hemagglutinating (HA) activity. Such hemagglutination was inhibited by a monoclonal anti-sigma 1 antibody previously shown to inhibit reovirus HA activity. It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact.