Dot diagrams as source documents for evaluations of test performance

Complete evaluations of test performance require data on many test results over many clinical states, not restricted to the traditional 2 X 2 table of two possible test results and two possible clinical states. Published reports on test performance often include dot diagrams, depicting many test results over many clinical states. From such dot diagrams, several methods may be used to obtain numerical data for quantitative evaluations of test performance. Dot diagrams, long used to depict multiple test results over multiple clinical states, can serve as source documents for quantitative evaluations of test performance.     

Cj1496c encodes a Campylobacter jejuni glycoprotein that influences invasion of human epithelial cells and colonization of the chick gastrointestinal tract

Campylobacter jejuni has an N-linked protein glycosylation pathway that is required for efficient cell invasion and chick gastrointestinal colonization by the microbe. In this study, we constructed insertion mutants of 22 putative glycoprotein genes and examined the ability of each to invade the human intestinal epithelial cell line INT-407. Among the mutants tested, one carrying an insertion in Cj1496c was defective for invasion into INT-407 cells; this defect was also observed in an in-frame deletion mutant of Cj1496c (delta Cj1496c). The delta Cj1496c mutant C. jejuni also showed a reduced ability to colonize chick ceca. Site-specific mutagenesis combined with Western blot analysis suggested that the Cj1496c protein is glycosylated at N73 and N169. However, the delta Cj1496c mutant expressing a nonglycosylated form of Cj1496c exhibited levels of invasion and colonization equivalent to those of the parent strain, suggesting that glycans are not directly involved in the function of Cj1496c.     

The common PPAR-gamma2 Pro12Ala variant is associated with greater insulin sensitivity

Several genetic variants of peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2) have been identified, among which Pro12Ala, a missense mutation in exon 2, is highly prevalent in Caucasian populations. Up to now, conflicting results with regard to the association between this mutation and complex traits, such as obesity, insulin sensitivity and Type 2 diabetes, have been reported. We investigated the influence of the Pro12Ala polymorphism of PPAR-gamma2 on insulin sensitivity in a large Italian population sample, n=1215, in whom extensive clinical and biochemical analyses were performed. To estimate the insulin sensitivity status, the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated; in the obese/overweight subjects an oral glucose tolerance test (OGTT) was also performed and the Matsuda insulin sensitivity index (ISI) calculated. The insulin secretion index (homeostasis model assessment of percent beta-cell function, HOMA-beta%) was utilized to evaluate beta-cell function. The effect of the Pro12Ala polymorphism on quantitative variables was tested using multiple linear regression analysis. X12Ala (either Pro12Ala or Ala12Ala) genotype was associated with significantly lower fasting insulin levels compared to Pro/Pro (P=0.01 after correction for multiple comparisons) in all subjects. Consistent with this finding, significantly lower HOMA-IR was observed in X12Ala carriers (P=0.013 after correction for multiple comparisons) in all cohort. Moreover, no significant interaction effect was observed between body mass index and X12Ala polymorphism and between gender and X12Ala polymorphism in modulating insulin sensitivity. Our observations substantially extend previous findings and demonstrated that X12Ala variant is significantly associated with greater insulin sensitivity.     

Factors associated with maintenance of wakefulness test mean sleep latency in patients with mild to moderate obstructive sleep apnoea and normal subjects

This study investigated the possible factors related to the Maintenance of Wakefulness Test (MWT) mean sleep latency. A second analysis explored the characteristics of subjects who had discrepant Epworth Sleepiness Scale (ESS) and MWT scores. A total of 151 subjects (110 mild to moderate obstructive sleep apnoea (OSA) patients and 41 control subjects) were recruited for the study. The subjects completed an overnight Polysomnography (PSG), MWT, cognitive, performance and vigilance tasks and answered self-report questionnaires on mood and sleepiness. A forward stepwise multiple regression was performed on MWT mean sleep latency. The predictor variables age (r = 0.28), subjective sleep history for 1 week prior to MWT (sleep diary; r = 0.19) and number of >4% SaO2 Dips during the PSG (r = -0.21) best explained the MWT results, but only accounted for 12.8% of the variance in the test. It was found that 33% of subjects had discrepant ESS and MWT scores. A new variable was created to analyse these subjects (MWT/ESS discrepancy score; MED). A forward stepwise multiple regression analysis found that depression, performance errors and sleep disordered breathing explained 13.4% of the variance in MED scores. The MWT is a complex behavioural test whose scores do not seem to have a very robust relationship with potential predictors and co-correlates. Further comprehensive study is needed if the test is to be used in a diagnostically meaningful way.     

The importance of the voxel size in clinical 1H spectroscopy of the human brain

It is demonstrated that it is possible to acquire two volume selective 1H NMR spectra of human brain in vivo, consisting of voxels of 1.5 X 1.5 X 1.5 cm3, within 14 min with a good S/N ratio. This is mainly achieved by the application of a PRESS sequence generating a spin-echo of the VOI at 135 ms in conjunction with the STABLE technique by which two spectra can be recorded in an interlaced mode. The Bo homogeneity over such small voxels is considerably higher than over larger voxels. With these methodological improvements it is possible to observe morphological heterogeneity of tumors. The results indicate that spectral changes seem to correlate with the metabolic state of the tumor rather than the tumor type. Additionally the spectrum of a patient with multiple sclerosis suggests that even differentiation between tumors and other lesions might not be possible.     

A new assay for quantifying human reticulocytes

The authors describe a new method for quantitative reticulocyte analysis. The assay uses a conventional clinical blood cell analyzer to size a patient's red blood cell neocyte population, which relates to the reticulocyte fraction in a linear fashion. Blood is layered atop Stractan (arabino-galactan polysaccharide) and centrifuged for 30 minutes at 1,500 X g. This density medium fractionation process enriches the Stractan layer with neocytes by up to 20-fold as determined by G6PD enzyme analysis. The mean corpuscular volume (MCV) of the red blood cells partitioning in the Stractan layer and the starting whole blood is then measured. The ratio of the two MCV measurements is then related to the reticulocyte percentage by a standard curve. In 93 patients, the derived MCV ratio was linearly correlated with manual reticulocyte counts (r = 0.96/slope = 0.99). Agreement of results obtained for single samples was within 0.2%. The assay's within-run and between-run precision is excellent (coefficient of variation less than 1%). The assay provides data on the percentage of reticulocytes in whole blood with an accuracy and precision that is at least 20 times greater than conventional microscopic technics.     

Identification of Immunogenic and Virulence-Associated Campylobacter jejuni Proteins

With the aim of identifying proteins important for host interaction and virulence, we have screened an expression library of NCTC 11168 Campylobacter jejuni genes for highly immunogenic proteins. A commercial C. jejuni open reading frame (ORF) library consisting of more than 1,600 genes was transformed into the Escherichia coli expression strain BL21(DE3), resulting in 2,304 clones. This library was subsequently screened for immunogenic proteins using antibodies raised in rabbit against a clinical isolate of C. jejuni; this resulted in 52 highly reactive clones representing 25 different genes after sequencing. Selected candidate genes were inactivated in C. jejuni NCTC 11168, and the virulence was examined using INT 407 epithelial cell line and motility, biofilm, autoagglutination, and serum resistance assays. These investigations revealed C. jejuni antigen 0034c (Cj0034c) to be a novel virulence factor and support the usefulness of the method. Further, several antigens were tested as vaccine candidates in two mouse models, in which Cj0034c, Cj0404, and Cj0525c resulted in a reduction of invasion in spleen and liver after challenge.     

高血压患者健康状况评估方法的研究

用生存质量量表与运动试验相结合的办法对39例血压正常者及51例高血压初期患者进行对比分析,比较各项测试指标,并用判别分析方法对两组人群进行评估分析.多项结果两组人存在显著性差异,建立的判别函数对两组区分率达到96.7%.本文的方法为评估高血压患者的健康状况提供了新的参考.     

Examination of Campylobacter jejuni Putative Adhesins Leads to the Identification of a New Protein,Designated FlpA,Required for Chicken Colonization

Campylobacter jejuni colonization of chickens is presumably dependent upon multiple surface-exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, major outer membrane protein, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of 97 C. jejuni isolates recovered from human, poultry, bovine, porcine, ovine, and canine sources by multilocus sequence typing (MLST) and examined their profile of putative adhesin-encoding genes by dot blot hybridization. To assess the individual contribution of each protein in bacterium-host cell adherence, the C. jejuni genes encoding the putative adhesins were disrupted by insertional mutagenesis. The phenotype of each mutant was judged by performing in vitro cell adherence assays with chicken LMH hepatocellular carcinoma epithelial cells and in vivo colonization assays with broiler chicks. MLST analysis indicated that the C. jejuni isolates utilized in this study were genetically diverse. Dot blot hybridization revealed that the C. jejuni genes encoding the putative adhesins, with the exception of capA, were conserved among the isolates. The C. jejuni CadF, CapA, Cj1279c, and Cj1349c proteins were found to play a significant role in the bacterium''s in vitro adherence to chicken epithelial cells, while CadF, PEB1, and Cj1279c were determined to play a significant role in the bacterium''s in vivo colonization of broiler chicks. Collectively, the data indicate that Cj1279c is a novel adhesin. Because Cj1279c harbors fibronectin type III domains, we designated the protein FlpA, for fibronectin-like protein A.Campylobacter jejuni is a gram-negative, spiral, microaerophilic bacterium, which is motile via a bipolar or unipolar flagellum. This organism is one of the leading bacterial causes of diarrhea in the United States and accounts for 5 to 14% of diarrhea worldwide (1). Experimental C. jejuni infections in humans have revealed that as few as 800 bacteria can cause human illness (4). Campylobacteriosis (C. jejuni-mediated gastroenteritis) generally occurs 2 to 5 days after ingestion of the bacterium and is a self-limiting infection. This disease is characterized by fever, nausea, malaise, abdominal pain, and loose-to-watery stools, which may contain blood and/or fecal leukocytes (5). C. jejuni infections can result in several serious sequelae, including Guillain-Barré syndrome, an acute autoimmune disease affecting the peripheral nervous system (35).C. jejuni infection frequently occurs through the ingestion of C. jejuni in undercooked chicken or from the consumption of food products cross-contaminated with raw poultry. This infection is linked to poultry due to the fact that by 2 to 3 weeks of age most commercial chickens become commensally colonized by as many as 10⁸ CFU of C. jejuni per g of cecal contents (33). Not surprisingly, Campylobacter organisms are frequently recovered from processed broiler carcasses (34). Bacterial adherence to host epithelial cells appears to be crucial for C. jejuni colonization of chickens, as it may prevent host-mediated mechanical forces such as peristalsis from clearing the bacterium. Previous work has revealed that one C. jejuni adhesin, termed CadF (Campylobacter adhesion to fibronectin [Fn]), is required to colonize Leghorn chickens (36).Researchers have identified a number of C. jejuni proteins that bind to cultured epithelial cells. These adhesive proteins include CadF, CapA, JlpA, major outer membrane protein (MOMP), and PEB1. Konkel et al. (18) identified CadF, which is a 37-kDa Fn-binding protein. CadF facilitates C. jejuni adherence to Fn, which is a ubiquitous ∼250-kDa glycoprotein found in the extracellular matrix and regions of cell-to-cell contact. A C. jejuni cadF mutant shows a 50% reduction in adhesion to human INT 407 intestinal cells compared to a C. jejuni wild-type isolate (28). Ashgar et al. (3) identified a putative autotransporter in silico, which was termed CapA, for Campylobacter adhesion protein A. C. jejuni capA is a contingency gene, in which expression of the functional protein is dependent upon frameshifts within a homopolymeric nucleotide tract located near the 5′ end of the capA coding region. A C. jejuni capA mutant demonstrated a reduction in adherence to human Caco-2 colorectal adenocarcinoma epithelial cells and a decrease in colonization efficiency of chickens. Jin et al. (13) identified JlpA (jejuni lipoprotein A), which is a 43-kDa protein. Disruption of jlpA reduces C. jejuni adherence to human HEp-2 epithelial cells by 18 to 19.4% relative to the wild-type strain. Moser et al. (29) reported that MOMP, encoded by porA, bound to INT 407 cells. The C. jejuni 43-kDa MOMP is also known to allow passage of hydrophilic molecules across the outer membrane and to provide structural stability to the outer membrane (6, 11). C. jejuni porA mutants have yet to be characterized, as mutations in porA are presumably lethal due to the critical structural and transport functions of the MOMP. Kervella et al. (15) characterized PEB1, a C. jejuni 28-kDa putative adhesin. Disruption of peb1A reduces C. jejuni adherence to human HeLa epithelial cells by 50- to 100-fold; such mutants also failed to colonize the intestinal tract of mice (32). In addition to the previously studied proteins listed above, Cj1279c and Cj1349c have been reported to contain Fn type III domains and to act as an Fn and fibrinogen-binding protein, respectively (http://www.microbesonline.org/; VIMSS identification no. 47155 and 47224, respectively). Due to the well-established role of CadF in C. jejuni cell adherence and chicken colonization, Cj1279c and Cj1349c were included in this study.C. jejuni binding to host cells and colonization are a multifactorial process dependent on motility, chemotaxis, and the synthesis of multiple adhesins (20). The goal of this study was to evaluate the contribution of the CadF, CapA, JlpA, PEB1, MOMP, Cj1279c, and Cj1349c proteins in C. jejuni-host cell interactions. Dot blot assays were used to assess the differences in the content of the putative adhesin-encoding genes among the C. jejuni isolates. To assess the roles of the putative adhesins in promoting host cell binding and host colonization, we generated C. jejuni adhesin mutants via insertional mutagenesis and performed in vitro adherence and in vivo chicken colonization assays. To our knowledge, this is the first time the roles of these proteins have been compared in a single genetic background and the first time that the functional role of many of these proteins has been examined in chickens (i.e., the natural host). We report that some of the C. jejuni proteins play a significant role in chicken colonization, while other proteins are not essential for colonization.     

Comparisons of Lasker's coefficient of relationship in a Venezuelan town in two different periods

A formula for the standard error of Lasker's coefficient of relationship Ri derived from isonymy is proposed, and used to test for differences in relationship in two groups of pairs of spouses from the town of Quibor in Venezuela sampled one century apart. From analysis of the relationship, it was possible to attribute population growth also to immigration. Further, the study of the values of Ri showed that the surnames belonging to the male line are more frequent and stable in this population, which is characterized by a predominantly agricultural activity. From the analysis of the coefficients of relationship, the population of Quibor is also classified as patrilocal.     

Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis

Campylobacter jejuni has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of C. jejuni transformants. A total of 1,065 C. jejuni transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.     

Gap junction beta 1 (<Emphasis Type="Italic">GJB1</Emphasis>) gene mutations in Italian patients with X-linked Charcot-Marie-Tooth disease

X-linked Charcot-Marie-Tooth disease (CMT1X) is a peripheral neuropathy transmitted in a dominant manner and caused by mutations in the Connexin 32 (Cx32) gene (GJB1, gap junction beta 1). Here we report the mutation analysis of the GJB1 gene in 76 subjects with possible CMT1 and absence of 17p11.2 duplication, and in 38 CMT2 patients without mutations in CMT2-associated-genes, selected from a cohort of 684 patients with peripheral sensory-motor neuropathy. The analysis was performed by direct sequencing of the coding sequence and exon/intron boundaries of the GJB1 gene. The mutation screening identified 22 mutations in GJB1, eight of which have not been previously published: six point mutations (c.50C > G, c.107T > A, c.545C > T, c.545C > G, c.548G > C, c.791G > T) and two deletions (c.84delC, c.573_581delCGTCTTCAT). The GJB1 mutation frequency (19.3%) and the clinical heterogeneity of our patients suggest searching for GJB1 mutations in all CMT cases without the 17p11.2 duplication, regardless of the gender of the proband, as well as in CMT2 patients with possible X-linked inheritance.     

Campylobacter jejuni Glycosylation Island Important in Cell Charge,Legionaminic Acid Biosynthesis,and Colonization of Chickens

Previously, we identified five genes (Cj1321 to Cj1326, of which Cj1325 and Cj1326 are a single gene) in the O-linked flagellin glycosylation island that are highly prevalent in Campylobacter jejuni isolates from chickens. We report mutagenesis, functional, and structural data to confirm that this locus, and Cj1324 in particular, has a significant contributory role in the colonization of chickens by C. jejuni. A motile ΔCj1324 mutant with intact flagella was considerably less hydrophobic and less able to autoagglutinate and form biofilms than the parent strain, 11168H, suggesting that the surface charge of flagella of Cj1324-deficient strains was altered. The physical and functional attributes of the parent were restored upon complementation. Structural analysis of flagellin protein purified from the ΔCj1324 mutant revealed the absence of two legionaminic acid glycan modifications that were present in the parent strain, 11168H. These glycoform modifications were shown to be prevalent in chicken isolates and confirm that differences in the highly variable flagellin glycosylation locus can relate to the strain source. The discovery of molecular mechanisms influencing the persistence of C. jejuni in poultry aids the rational design of approaches to control this problematic pathogen in the food chain.Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide (7). Infection can cause symptoms including abdominal pain and fever with watery to bloody diarrhea (54). Occasionally, postinfectious sequelae follow C. jejuni infection and include reactive arthritis and Guillain-Barré syndrome (8). Recently, C. jejuni has been associated with immunoproliferative small intestine disease, which is a rare type of mucosa-associated lymphoid tissue lymphoma (31). The main source of transmission through the food chain is the consumption and handling of contaminated poultry, but the underlying reasons why chickens are particularly susceptible to colonization by C. jejuni are unknown (15). C. jejuni has also been recovered from nonavian livestock, unpasteurized milk, and contaminated water (7). The socioeconomic burden of this pathogen means that it is imperative that ways of reducing the levels of C. jejuni in the food chain, particularly poultry, are investigated.The glycosylation of flagellin in a number of gram-negative pathogenic bacteria, including Pseudomonas aeruginosa, Helicobacter pylori, and Aeromonas spp., is increasingly recognized as playing significant roles (2, 24, 32, 43, 49). Glycosylation modifications have been shown to influence the cell''s immunogenicity, interaction with eukaryotic cells, and host cell specificity. Aeromonads are waterborne bacteria that can cause disease in fish, reptiles, and amphibians. Mesophilic aeromonads are important human pathogens causing gastrointestinal infections and, in severe cases, wound disease and septicemia in healthy and immunocompromised patients (63). Flagella of the mesophilic aeromonad Aeromonas caviae have been shown to be glycosylated (43) with a derivative of pseudaminic acid (50). In the plant pathogen Pseudomonas syringae pv. glycinea, the mutation of three genes located in a flagellin glycosylation island results in alterations to host specificity (61). Mutants of P. syringae pv. glycinea fail to cause symptoms in the normal host, soybean plants, but can grow on nonhost tobacco leaves, causing symptom-like changes on leaves. Takeuchi et al. proposed that the posttranslational modification of flagellin may be an adaptation of the bacterium to avoid recognition by host defenses (61). In P. aeruginosa strain PAK, a flagellin glycosylation island comprising 14 genes was discovered and shown to cause glycosylation exclusively for P. aeruginosa isolates expressing a-type flagellin (2). Further studies have shown that there appears to be variation in the glycosylation islands of strains containing the a-type flagellin (4). A glycosylation island comprising four genes in the type b flagellin strain P. aeruginosa PAO has been found. When a mutant unable to glycosylate flagellin was tested in a murine model of burn wound infection, it exhibited a reduction in virulence compared to that of the wild type (3). Thus, it appears that in P. aeruginosa different glycoforms on the flagellin are required for the colonization of different hosts or environments and that these glycoforms may provide the bacterium with a specific survival advantage.We recently examined 111 strains of C. jejuni, including human, chicken, bovine, ovine, and environmental isolates, using comparative phylogenomics (whole-genome comparisons of microbes using DNA microarrays combined with Bayesian-based phylogenies) (10). Isolates fell into two distinct clades, which based on the origins of the isolates were defined as livestock-associated and non-livestock-associated clades. Over 40 genes were identified as being significantly prevalent in either of the clades. Among these was a set of six genes, Cj1321, Cj1322, Cj1323, Cj1324, Cj1325, and Cj1326 (as identified in the initial annotation by Parkhill et al. for the original sequenced C. jejuni strain, NCTC11168 [42]), that lie within a region of the genome encoding the flagellin O-linked glycosylation system. Thus, although genes Cj1321 to Cj1326 are located within a region of the genome which has variability, they are conserved among some C. jejuni strains that are often associated with livestock. Microarray data have shown that the six genes are all transcribed in the same orientation, but it is unknown if they are an operon (N. Dorrell and B. W. Wren, unpublished data). NCTC11168 has since been reannotated, and as a result, Cj1325 and Cj1326 are now considered to be one gene, hereinafter referred to as Cj1325/6 (22). Previous BLAST analyses have shown that the Cj1321 protein has amino acid similarity to many bacterial acetyl transferases, both Cj1322 and Cj1323 proteins are similar to hydroxyacyl dehydrogenases, and the product of Cj1324 is similar to WbpG, a protein involved in lipopolysaccharide synthesis in many bacteria.In C. jejuni NCTC11168 (the original sequenced strain, found in the livestock clade), the O-linked flagellar glycosylation system is thought to consist of a cluster of approximately 50 genes (Cj1293 to Cj1342) adjacent to flaA and flaB which encode the structural flagellin proteins (42). The full glycan structure(s) in NCTC11168 (and most other strains associated with livestock) is unknown, but given the considerably larger size of the O-linked glycosylation loci in the livestock-associated strains than in the non-livestock-associated strains, it is likely that the livestock-associated strains may have additional modifications to the pseudaminic acid basic structure, as well as other unique glycan moieties, compared to those of the non-livestock-associated strains. The flagellin O-linked glycosylation locus in C. jejuni 81-176 (a frequently studied human strain found in the non-livestock-associated clade) is far simpler than that in C. jejuni NCTC11168, comprising just 26 genes (21). Two modifications predominantly decorate FlaA and FlaB of strain 81-176, the nine-carbon sugar pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid [Pse5Ac7Ac]) and an acetamidino form of pseudaminic acid, 5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (Pse5Ac7Am). Derivatives of Pse5Ac7Am also decorate the flagellin of 81-176 in minor quantities (34, 37, 62). Genetic analysis of 81-176 showed that pse genes are involved in the biosynthesis of pseudaminic acid and its derivatives (21, 37, 62). More recently, the full biosynthetic pathway for pseudaminic acid was determined; in a six-step reaction, UDP-N-acetylglucosamine (UDP-GlcNAc) is converted to pseudaminic acid through the actions of PseB/Cj1293, PseC/Cj1294, PseH/Cj1313, PseG/Cj1312, PseI/Cj1317, and PseF/Cj1311 proteins (The Cj designations refer to predicted coding sequences in C. jejuni NCTC11168) (11, 19, 21, 35, 52, 62).The most detailed analysis of the flagellin O-linked glycosylation locus has been undertaken with C. jejuni strain 81-176 and the related species Campylobacter coli (strain VC167) (34). Structural studies of the flagellum modifications of C. coli VC167 revealed that in addition to Pse5Ac7Ac, acetamidino and N-methylacetimidoyl derivatives of legionaminic acid [5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-nonulosonic acid (Leg5Am7Ac) and 5-E/Z-N-(N-methlyacetimidoyl)-7-acetamidino-3,5,7,9-tetradeoxy-d-galacto-nonulosonic acid (Leg5AmNMe7Ac), respectively] decorate the C. coli flagellin, the first demonstration of a legionaminic acid derivative modification of bacterial flagellin (36). Biosynthesis of these legionaminic acid derivatives involves a distinct pathway encoded by the posttranslational modification (ptm) genes (34, 36). Although the precise pathway for the production of legionaminic acid has yet to be determined, tentative functions have been assigned which have identified PtmA to PtmH to be required for biosynthesis (PtmA, PtmB, PtmC, PtmD, PtmE, PtmF, PtmG, and PtmH are equivalent to the Cj1332, Cj1331, Cj1327, Cj1328, Cj1329, Cj1330, Cj1324, and Cj1325/6 proteins, respectively) (36). This ptm pathway is absent in C. jejuni strain 81-176. PtmG and PtmH from C. coli VC167 show 86 and 76% amino acid sequence similarity, respectively, to two hypothetical proteins, the Cj1324 and Cj1325/6 proteins of C. jejuni NCTC11168. The enzyme(s) involved in the attachment of glycan(s) to the flagellin protein of Campylobacter strains and the consensus sequence for the O-linked glycosylation process have yet to be identified.In C. jejuni strain 81-176, glycosylation of flagellin has been shown to be necessary for the assembly of flagella and subsequent motility (19). There is extensive polymorphism in the C. jejuni O-linked glycosylation cluster, suggesting that selective pressure may cause the bacterium to alter surface antigens in attempts to evade the host immune defenses (59). Evidence supporting this possibility is demonstrated by comparing the glycan moieties of the flagella of C. jejuni 81-176 and C. coli VC167, as these strains produce unique modifications on their flagella which affect serospecificity (34).Given the diversity of the O-linked glycosylation system in C. jejuni and the prevalence of the locus of Cj1321 to Cj1325/6 in chicken isolates, we hypothesized that these genes may be important for the abilities of some C. jejuni strains to colonize poultry and that colonization may be mediated through structural and surface charge changes in the glycan that modifies the flagellin. In this study, we demonstrate that Cj1324 is involved in the biosynthesis of two novel legionaminic acid modifications found on the flagellin of strain 11168H. The presence of these modifications affects autoagglutination, cell charge, and the efficiency with which C. jejuni 11168H colonizes chickens.     

Development of the University of Pennsylvania Smell Identification Test: a standardized microencapsulated test of olfactory function

The development of the first standardized "scratch 'n sniff" olfactory test is described. Over 1600 subjects participated in five experiments. In Experiment 1, 50 microencapsulated odorants were rated as to their intensity, pleasantness, irritation, coolness, and familiarity, and two procedures for releasing them were compared. In Experiment 2, the results of the first experiment and other data were used in the development of the test, which was administered to a large number of subjects. Using multiple regression analysis, scores on this test were shown to be significantly related to the subjects' gender, ethnic background, and smoking behavior. Average test scores decreased as a function of age, with the greatest decline occurring between the sixth and tenth decades of life. These age-related changes were not correlated with scores on the Wechsler Memory Scale. Women performed better than men within all age categories. In Experiment 3, the test was shown to differentiate between subjects with known olfactory disorders (e.g., Kallmann's syndrome; Korsakoff's syndrome) and normal controls, and to reliably detect persons instructed to feign total anosmia. In Experiment 4, the test-retest reliability was established (6-month interval; r = 0.918, p less than 0.001), and in Experiment 5 the test was shown to correlate thresholds with odor detection (r = -0.794, p less than 0.001). This self-administratered test now makes it possible to rapidly and accurately assess general olfactory function in the laboratory, clinic, or through the mail without complex equipment or space-consuming stores of chemicals.     

Screening for the fragile X syndrome among mentally retarded males by hair root analysis

A noninvasive antibody test was used to identify male fragile X patients in special education schools, on the basis of the lack of FMRP in hair roots. We studied 300 males with mental retardation of unknown cause attending special schools. Patients were divided into two groups, based on the scores according to a fragile X check list (Group 1 /= 10 points). Group 2 consists of 51 males and only 5 males in this group showed no FMRP expression in hair roots within the abnormal range (91%). Fragile X diagnosis in these cases was confirmed by DNA analysis. None of the males scoring more than 10 on the check list was diagnosed positive for the fragile X syndrome using DNA analysis. With our antibody test on hair roots we did not detect a fragile X patient in Group 1. The FMRP antibody test on hair roots is suitable in a screening program for the fragile X syndrome among mentally retarded males attending special education schools.     

DR2-positive monozygotic twins discordant for narcolepsy

Narcolepsy runs in families, and recent research has revealed the human leukocyte antigen (HLA) DR2 to be a genetic marker closely associated with the disease. But, as indicated by family studies, other factors contribute to the pathogenesis of narcolepsy. The investigation of monozygotic twins is the most specific research tool for distinguishing between a multigenetic and a multifactorial pathogenetic model. We present clinical and sleep polygraphic data from two pairs of monozygotic twins, and in addition, from some of their first-degree relatives. In both pairs only one twin suffered from the clinical symptoms of narcolepsy/cataplexy. Only in these subjects did night sleep recordings and a multiple sleep latency test reveal both multiple sleep onset rapid-eye-movement periods (SOREMPs) and short mean sleep onset latencies. However, in two of the asymptomatic, HLA DR2+ relatives, short mean sleep onset latencies during the multiple sleep latency test (MSLT) were observed, and one, HLA DR2- relative showed REM sleep two times during the MSLT. Our results strongly favor a multifactorial pathogenetic model for narcolepsy.     

The ratio of waist-to-hip circumference, plasma insulin level, and glucose intolerance as independent predictors of the HDL2 cholesterol level in older adults

High plasma levels of HDL2, a subfraction of high-density lipoprotein (HDL) cholesterol, are associated with a reduced risk of coronary heart disease. To investigate the characteristics related to HDL2 cholesterol levels, we measured lipoprotein levels and several metabolic and anthropometric variables in 146 healthy subjects (77 men and 69 women) in the seventh decade of life. The level of HDL2 cholesterol was inversely correlated with the ratio of the waist-to-hip circumference (r = -0.335 for men; r = -0.370 for women; P less than 0.01) and the plasma insulin level (r = -0.400 for men; r = -0.398 for women; P less than 0.001). In a multiple regression model including both sexes, 41 percent of the variance in the HDL2 level was explained by the combined effect of the waist-to-hip ratio (P less than 0.0001), the plasma insulin level (P = 0.0003), and the degree of glucose tolerance indicated by the integrated area under the plasma glucose curve after an oral glucose-tolerance test (P = 0.05). The body-mass index, total percentage of body fat, maximal oxygen uptake, diet, and sex were not significant predictors of the HDL2 level when added to this model, whereas the original variables remained significant predictors. The HDL2 cholesterol level in subjects at the 25th percentile for waist-to-hip ratio was 153 percent of that in subjects at the 75th percentile. We conclude that HDL2 levels are inversely correlated with truncal fat, plasma insulin levels, and the presence of glucose intolerance and are not independently associated with sex or total body fat.