The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

Dividing cells from persons with Bloom's syndrome, an autosomal recessive disorder of growth, exhibit increased numbers of chromatid breaks and rearrangements. A highly characteristic feature of the chromosome instability in this syndrome is the tendency for exchanges to occur between chromatids of homologous chromosomes at homologous sites. In the present experiments, a cytogenetic technique by which the sister chromatids of a metaphase chromosome are stained differentially has been used to demonstrate a striking and possibly specific, but hitherto unrecognized, increase in the frequency with which sister chromatids also exchange segments. The cells were grown in bromodeoxyuridine and stained with 33258 Hoechst and Giemsa. Whereas phytohemagglutinin-stimulated lymphocytes from normal controls had a mean of 6.9 sister chromatid exchanges per metaphase (range 1-14), those from persons with Bloom's syndrome had a mean of 89.0 (range 45-162). Normal frequencies of sister chromatid exchanges were found in cells heterozygous for the Bloom's syndrome gene, and also in cells either homozygous or heterozygous for the genes of the Louis-Bar (ataxia telangiectasia) syndrome and Fanconi's anemia, two other rare disorders characterized by chromosome instability. In a differentially stained chromatid interchange configuration discovered during the study, it was possible to determine the new distribution of both sister and non-sister-but-homologous chromatids that had resulted from numerous exchanges. By following shifts in the pattern of staining from chromatid to chromatid, visual evidence was obtained that the quadriradial configurations long recognized as characteristic of Bloom's syndrome represent exchanges between homologous chromosomes, apparently at homologous points. We postulate that the increase in the frequency of exchanges between nonsister-but-homologous chromatids and those between sister chromatids in Bloom's syndrome represents aspects of one and the same disturbance. A study of this phenomenon in relation to the clinical features of Bloom's syndrome may be helpful eventually in understanding the biological significance of chromatid exchange in somatic cells.

BETA-Lactamases (EC. 3.5.2.6) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of beta-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate beta-lactamase in mutants previously reported to lack the enzyme. R that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R1 and RGN14, with identical isoelectric focusing patterns have the same biochemical properties. Chromosomal and R-factor-mediated beta-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing. R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of beta-lactamases carried by R factors.

Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5-20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after approximately 1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only approximately 20-fold over this period, whereas donor-genotype endothelial cells expanded approximately 1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts.

OBJECTIVE

To analyze the long-term outcome of patients included in the Lymphome Non Hodgkinien study 98-5 (LNH98-5) comparing cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) to rituximab plus CHOP (R-CHOP) in elderly patients with diffuse large B-cell lymphoma.

METHODS

LNH98-5 was a randomized study that included 399 previously untreated patients, age 60 to 80 years, with diffuse large B-cell lymphoma. Patients received eight cycles of classical CHOP (cyclophosphamide 750 mg/m(2), doxorubicin 50 mg/m(2), vincristine 1.4 mg/m(2), and prednisone 40 mg/m(2) for 5 days) every 3 weeks. In R-CHOP, rituximab 375 mg/m(2) was administered the same day as CHOP. Survivals were analyzed using the intent-to-treat principle.

RESULTS

Median follow-up is 5 years at present. Event-free survival, progression-free survival, disease-free survival, and overall survival remain statistically significant in favor of the combination of R-CHOP (P = .00002, P < .00001, P < .00031, and P < .0073, respectively, in the log-rank test). Patients with low-risk or high-risk lymphoma according to the age-adjusted International Prognostic Index have longer survivals if treated with the combination. No long-term toxicity appeared to be associated with the R-CHOP combination.

CONCLUSIONS

Using the combination of R-CHOP leads to significant improvement of the outcome of elderly patients with diffuse large B-cell lymphoma, with significant survival benefit maintained during a 5-year follow-up. This combination should become the standard for treating these patients.

The rotational positioning of DNA about the histone octamer appears to be determined by certain sequence-dependent modulations of DNA structure. To establish the detailed nature of these interactions, we have analysed the sequences of 177 different DNA molecules from chicken erythrocyte core particles. All variations in the sequence content of these molecules, which may be attributed to sequence-dependent preferences for DNA bending, correlate well with the detailed path of the DNA as it wraps around the histone octamer in the crystal structure of the nucleosome core. The sequence-dependent preferences that correlate most closely with the rotational orientation of the DNA, relative to the surface of the protein, are of two kinds: ApApA/TpTpT and ApApT/ApTpT, the minor grooves of which face predominantly in towards the protein; and also GpGpC/GpCpC and ApGpC/GpCpT, whose minor grooves face outward. Fourier analysis has been used to obtain fractional variations in occurrence for all ten dinucleotide and all 32 trinucleotide arrangements. These sequence preferences should apply generally to many other cases of protein-DNA recognition, where the DNA wraps around a protein. In addition, it is observed that long runs of homopolymer (dA) X (dT) prefer to occupy the ends of core DNA, five to six turns away from the dyad. These same sequences are apparently excluded from the near-centre of core DNA, two to three turns from the dyad. Hence, the translational positioning of any single histone octamer along a DNA molecule of defined sequence may be strongly influenced by the placement of (dA) X (dT) sequences. It may also be influenced by any aversion of the protein for sequences in the "linker" region, the sequence content of which remains to be determined.

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on "rigid" glass or "stiff" gels than on "soft" gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.

Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended ("off-target") consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3' UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with dependence on siRNA concentration similar to that of silencing of the target transcript. Thus, short stretches of sequence complementarity to the siRNA or shRNA seed region are key to the silencing of unintended transcripts.

This paper presents a simple and efficient method for oligonucleotide-directed mutagenesis using vectors derived from single-stranded phage. This modification of our previously published procedure (Zoller and Smith, 1982) features the use of two primers, one of which is a standard M13 sequencing primer and the other is the mutagenic oligonucleotide. Both primers are simultaneously annealed to single-stranded template DNA, extended by DNA polymerase I (large fragment), and ligated together to form a mutant wild-type gapped heteroduplex. Escherichia coli is transformed directly with this DNA; the isolation of covalently closed circular DNA as in our previous report is not necessary. Mutants are identified by plaque lift hybridization using the mutagenic oligonucleotide as a probe. As an example of the method, a heptadecanucleotide was used to create a T----G transversion in the MATa gene of Saccharomyces cerevisiae cloned into the vector M13mp5. The efficiency of mutagenesis was approximately 50%. Production of the desired mutation was verified by DNA sequencing. The same procedure has been used without modification to create insertions of restriction sites as well as specific deletions of 500 bases.

BACKGROUND

Overweight in adults is associated with increased morbidity and mortality. In contrast, the long-term effect of overweight in adolescence on morbidity and mortality is not known.

METHODS

We studied the relation between overweight and morbidity and mortality in 508 lean or overweight adolescents 13 to 18 years old who participated in the Harvard Growth Study of 1922 to 1935. Overweight adolescents were defined as those with a body-mass index that on two occasions was greater than the 75th percentile in subjects of the same age and sex in a large national survey. Lean adolescents were defined as those with a body-mass index between the 25th and 50th percentiles. Subjects who were still alive were interviewed in 1988 to obtain information about their medical history, weight, functional capacity, and other risk factors. For those who had died, information on the cause of death was obtained from death certificates.

RESULTS

Overweight in adolescent subjects was associated with an increased risk of mortality from all causes and disease-specific mortality among men, but not among women. The relative risks among men were 1.8 (95 percent confidence interval, 1.2 to 2.7; P = 0.004) for mortality from all causes and 2.3 (95 percent confidence interval, 1.4 to 4.1; P = 0.002) for mortality from coronary heart disease. The risk of morbidity from coronary heart disease and atherosclerosis was increased among men and women who had been overweight in adolescence. The risk of colorectal cancer and gout was increased among men and the risk of arthritis was increased among women who had been overweight in adolescence. Overweight in adolescence was a more powerful predictor of these risks than overweight in adulthood.

CONCLUSIONS

Overweight in adolescence predicted a broad range of adverse health effects that were independent of adult weight after 55 years of follow-up.

Spontaneous discharge of norepinephrine-containing locus coeruleus (NE-LC) neurons was examined during the sleep-walking cycle (S-WC) in behaving rats. Single unit and multiple unit extracellular recordings yielded a consistent set of characteristic discharge properties. (1) Tonic discharge co-varied with stages of the S-WC, being highest during waking, lower during slow wave sleep, and virtually absent during paradoxical sleep. (2) Discharge anticipated S-WC stages as well as phasic cortical activity, such as spindles, during slow wave sleep. (3) Discharge decreased within active waking during grooming and sweet water consumption. (4) Bursts of impulses accompanied spontaneous or sensory-evoked interruptions of sleep, grooming, consumption, or other such ongoing behavior. (5) These characteristic discharge properties were topographically homogeneous for recordings throughout the NE-LC. (6) Phasic robust activity was synchronized markedly among neurons in multiple unit populations. (7) Field potentials occurred spontaneously in the NE-LC and were synchronized with bursts of unit activity from the same electrodes. (8) Field potentials became dissociated from unit activity during paradoxical sleep, exhibiting their highest rates in the virtual absence of impulses. These results are generally consistent with previous proposals that the NE-LC system is involved in regulating cortical and behavioral arousal. On the basis of the present data and those described in the following report (Aston-Jones, G., and F. E. Bloom (1981) J. Neurosci.1: 887-900), we conclude that these neurons may mediate a specific function within the general arousal framework. In brief, the NE-LC system may globally bias the responsiveness of target neurons and thereby influence overall behavioral orientation.

BACKGROUND

Recent research has indicated that autism is not a discrete disorder and that family members of autistic probands have an increased likelihood of exhibiting autistic symptoms with a wide range of severity, often below the threshold for a diagnosis of an autism spectrum disorder.

OBJECTIVE

To examine the distribution and genetic structure of autistic traits in the general population using a newly established quantitative measure of autistic traits, the Social Responsiveness Scale (formerly known as the Social Reciprocity Scale).

METHODS

The sample consisted of 788 pairs of twins aged 7 to 15 years, randomly selected from the pool of participants in a large epidemiologic study (the Missouri Twin Study). One parent of each pair of twins completed the Social Responsiveness Scale on each child. The data were subjected to structural equation modeling.

RESULTS

Autistic traits as measured by the Social Responsiveness Scale were continuously distributed and moderately to highly heritable. Levels of severity of autistic traits at or above the previously published mean for patients with pervasive developmental disorder not otherwise specified were found in 1.4% of boys and 0.3% of girls. Structural equation modeling revealed no evidence for the existence of sex-specific genetic influences, and suggested specific mechanisms by which females may be relatively protected from vulnerability to autistic traits.

CONCLUSIONS

These data indicate that the social deficits characteristic of autism spectrum disorders are common. Given the continuous distribution of these traits, it may be arbitrary where cutoffs are made between research designations of being "affected" vs "unaffected" with a pervasive developmental disorder. The genes influencing autistic traits appear to be the same for boys and girls. Lower prevalence (and severity) of autistic traits in girls may be the result of increased sensitivity to early environmental influences that operate to promote social competency.

Many bacteria live only within animal cells and infect hosts through cytoplasmic inheritance. These endosymbiotic lineages show distinctive population structure, with small population size and effectively no recombination. As a result, endosymbionts are expected to accumulate mildly deleterious mutations. If these constitute a substantial proportion of new mutations, endosymbionts will show (i) faster sequence evolution and (ii) a possible shift in base composition reflecting mutational bias. Analyses of 16S rDNA of five independently derived endosymbiont clades show, in every case, faster evolution in endosymbionts than in free-living relatives. For aphid endosymbionts (genus Buchnera), coding genes exhibit accelerated evolution and unusually low ratios of synonymous to nonsynonymous substitutions compared to ratios for the same genes for enterics. This concentration of the rate increase in nonsynonymous substitutions is expected under the hypothesis of increased fixation of deleterious mutations. Polypeptides for all Buchnera genes analyzed have accumulated amino acids with codon families rich in A+T, supporting the hypothesis that substitutions are deleterious in terms of polypeptide function. These observations are best explained as the result of Muller's ratchet within small asexual populations, combined with mutational bias. In light of this explanation, two observations reported earlier for Buchnera, the apparent loss of a repair gene and the overproduction of a chaperonin, may reflect compensatory evolution. An alternative hypothesis, involving selection on genomic base composition, is contradicted by the observation that the speedup is concentrated at nonsynonymous sites.

We describe a full structural model for amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)), based on numerous constraints from solid state NMR and electron microscopy. This model applies specifically to fibrils with a periodically twisted morphology, with twist period equal to 120 +/- 20 nm (defined as the distance between apparent minima in fibril width in negatively stained transmission electron microscope images). The structure has threefold symmetry about the fibril growth axis, implied by mass-per-length data and the observation of a single set of (13)C NMR signals. Comparison with a previously reported model for Abeta(1-40) fibrils with a qualitatively different, striated ribbon morphology reveals the molecular basis for polymorphism. At the molecular level, the 2 Abeta(1-40) fibril morphologies differ in overall symmetry (twofold vs. threefold), the conformation of non-beta-strand segments, and certain quaternary contacts. Both morphologies contain in-register parallel beta-sheets, constructed from nearly the same beta-strand segments. Because twisted and striated ribbon morphologies are also observed for amyloid fibrils formed by other polypeptides, such as the amylin peptide associated with type 2 diabetes, these structural variations may have general implications.

Prenatal famine in humans has been associated with various later-life consequences, depending on the gestational timing of the insult and the sex of the exposed individual. Epigenetic mechanisms have been proposed to underlie these associations. Indeed, animal studies and our early human data on the imprinted IGF2 locus indicated a link between prenatal nutritional and DNA methylation. However, it remains unclear how common changes in DNA methylation are and whether they are sex- and timing-specific paralleling the later-life consequences of prenatal famine exposure. To this end, we investigated the methylation of 15 loci implicated in growth and metabolic disease in individuals who were prenatally exposed to a war-time famine in 1944-45. Methylation of INSIGF was lower among individuals who were periconceptionally exposed to the famine (n = 60) compared with their unexposed same-sex siblings (P = 2 x 10(-5)), whereas methylation of IL10, LEP, ABCA1, GNASAS and MEG3 was higher (all P < 10(-3)). A significant interaction with sex was observed for INSIGF, LEP and GNASAS. Next, methylation of eight representative loci was compared between 62 individuals exposed late in gestation and their unexposed siblings. Methylation was different for GNASAS (P = 1.1 x 10(-7)) and, in men, LEP (P = 0.017). Our data indicate that persistent changes in DNA methylation may be a common consequence of prenatal famine exposure and that these changes depend on the sex of the exposed individual and the gestational timing of the exposure.

BACKGROUND

Huntington's disease (HD) is a fatal and progressive neurodegenerative disease that is accompanied by involuntary movements, cognitive dysfunction, and psychiatric symptoms. Although progressive striatal degeneration is known to occur, little is known about how the disease affects the cortex, including which cortical regions are affected, how degeneration proceeds, and the relationship of the cortical degeneration to clinical symptoms. The cortex has been difficult to study in neurodegenerative diseases primarily because of its complex folding patterns and regional variability; however, an understanding of how the cortex is affected by the disease may provide important new insights into it.

METHODS

Novel automated surface reconstruction and high-resolution MR images of 11 patients with HD and 13 age-matched subjects were used to obtain cortical thickness measurements. The same analyses were performed on two postmortem brains to validate these methods.

RESULTS

Regionally specific heterogeneous thinning of the cortical ribbon was found in subjects with HD. Thinning occurred early, differed among patients in different clinical stages of disease, and appeared to proceed from posterior to anterior cortical regions with disease progression. The sensorimotor region was statistically most affected. Measurements performed on MR images of autopsy brains analyzed similarly were within 0.25 mm of those obtained using traditional neuropathologic methods and were statistically indistinguishable.

CONCLUSIONS

The authors propose that the cortex degenerates early in disease and that regionally selective cortical degeneration may explain the heterogeneity of clinical expression in HD. These measures might provide a sensitive prospective surrogate marker for clinical trials of neuroprotective medications.

Involvement of the hippocampus and caudate nucleus in place and response learning was examined by functionally inactivating these brain regions bilaterally with infusions of lidocaine. Rats were trained to approach a consistently baited arm in a cross-maze from the same start box (four trials/day/14 total days). On Days 8 and 16 a single probe trial was given, in which rats were placed in the start box opposite that used in training and allowed to approach a maze arm. Three minutes prior to the probe trial, rats received bilateral injections of either saline or a 2% lidocaine solution (in order to produce neural inactivation) into either the dorsal hippocampus or dorsolateral caudate nucleus. On the probe trials, rats which entered the baited maze arm (i.e., approached the place where food was located during training) were designated place learners, and rats which entered the unbaited maze arm (i.e., made the same turning response as during training) were designated response learners. Saline-treated rats displayed place learning on the Day 8 probe trial and response learning on the Day 16 probe trial, indicating that with extended training there is a shift in learning mechanisms controlling behavior. Rats given lidocaine injections into the hippocampus showed no preference for place or response learning on the Day 8 probe trial, but displayed response learning on the Day 16 probe trial, indicating a blockade of place learning following inactivation of the hippocampus. Rats given lidocaine injections into the caudate nucleus displayed place learning on both the Day 8 and the Day 16 probe trials, indicating a blockade of response learning following inactivation of the caudate nucleus. The findings indicate: (1) the hippocampus and caudate nucleus selectively mediate expression of place and response learning, respectively (2), in a visually cued extramaze environment, hippocampal-dependent place learning is acquired faster than caudate-dependent response learning, and (3) when animals shift to caudate-dependent response learning with extended training, the hippocampal-based place representation remains intact.

Three strains of enteropathogenic Escherichia coli (EPEC), originally isolated from humans and previously shown to cause diarrhea in human volunteers by unknown mechanisms, and one rabbit EPEC strain were shown to attach intimately to and efface microvilli and cytoplasm from intestinal epithelial cells in both the pig and rabbit intestine. The attaching and effacing activities of these EPEC were demonstrable by light microscopic examination of routine histological sections and by transmission electron microscopy. It was suggested that intact colostrum-deprived newborn pigs and ligated intestinal loops in pigs and rabbits may be useful systems to detect EPEC that have attaching and effacing activities and for studying the pathogenesis of such infections. The lesions (attachment and effacement) produced by EPEC in these systems were multifocal, with considerable animal-to-animal variation in response to the same strain of EPEC. The EPEC strains also varied in the frequency and extent of lesion production. For example, three human EPEC strains usually caused extensive lesions in rabbit intestinal loops, whereas two other human EPEC strains usually did not produce lesions in this system.

Two genomic clones (lambda Ubi-1 and lambda Ubi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25 degrees C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5' untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5' flanking region and the entire 5' untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.

In a variety of plant species, the development of necrotic lesions in response to pathogen infection leads to induction of generalized disease resistance in uninfected tissues. A well-studied example of this "immunity" reaction is systemic acquired resistance (SAR) in tobacco. SAR is characterized by the development of a disease-resistant state in plants that have reacted hypersensitively to previous infection by tobacco mosaic virus. Here, we show that the onset of SAR correlates with the coordinate induction of nine classes of mRNAs. Salicylic acid, a candidate for the endogenous signal that activates the resistant state, induces expression of the same "SAR genes." A novel synthetic immunization compound, methyl-2,6-dichloroisonicotinic acid, also induces both resistance and SAR gene expression. These observations are consistent with the hypothesis that induced resistance results at least partially from coordinate expression of these SAR genes. A model is presented that ties pathogen-induced necrosis to the biosynthesis of salicylic acid and the induction of SAR.

BACKGROUND

The McCune-Albright syndrome is a sporadic disease characterized by polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunction of multiple endocrine glands. These manifestations may be explained by a somatic mutation in affected tissues that results in activation of the signal-transduction pathway generating cyclic AMP (cAMP). We analyzed DNA from tissues of patients with the McCune-Albright syndrome for the presence of activating mutations of the gene for the alpha subunit of the G protein (Gs alpha) that stimulates cAMP formation.

METHODS

Genomic DNA fragments encompassing regions (exons 8 and 9) previously found to contain activating missense mutations of the Gs alpha gene (gsp mutations) in sporadically occurring pituitary tumors were amplified in tissues from four patients with the McCune-Albright syndrome by the polymerase chain reaction. The amplified DNA was analyzed for mutations by denaturing gradient gel electrophoresis and allele-specific oligonucleotide hybridization.

RESULTS

We detected one of two activating mutations within exon 8 of the Gs alpha gene in tissues from all four patients, including affected endocrine organs (gonads, adrenal glands, thyroid, and pituitary) and tissues not classically involved in the McCune-Albright syndrome. In two of the patients, histidine was substituted for arginine at position 201 of Gs alpha, and in the other two patients cysteine was substituted for the same arginine residue. In each patient the proportion of cells affected varied from tissue to tissue. In two endocrine organs, the highest proportion of mutant alleles was found in regions of abnormal cell proliferation.

CONCLUSIONS

Mutations within exon 8 of the Gs alpha gene that result in increased activity of the Gs protein and increased cAMP formation are present in various tissues of patients with the McCune-Albright syndrome. Somatic mutation of this gene early in embryogenesis could result in the mosaic population of normal and mutant-bearing tissues that may underlie the clinical manifestations of this disease.

BACKGROUND

Hand hygiene prevents cross infection in hospitals, but compliance with recommended instructions is commonly poor. We attempted to promote hand hygiene by implementing a hospital-wide programme, with special emphasis on bedside, alcohol-based hand disinfection. We measured nosocomial infections in parallel.

METHODS

We monitored the overall compliance with hand hygiene during routine patient care in a teaching hospital in Geneva, Switzerland, before and during implementation of a hand-hygiene campaign. Seven hospital-wide observational surveys were done twice yearly from December, 1994, to December, 1997. Secondary outcome measures were nosocomial infection rates, attack rates of methicillin-resistant Staphylococcus aureus (MRSA), and consumption of handrub disinfectant.

RESULTS

We observed more than 20,000 opportunities for hand hygiene. Compliance improved progressively from 48% in 1994, to 66% in 1997 (p<0.001). Although recourse to handwashing with soap and water remained stable, frequency of hand disinfection substantially increased during the study period (p<0.001). This result was unchanged after adjustment for known risk factors of poor adherence. Hand hygiene improved significantly among nurses and nursing assistants, but remained poor among doctors. During the same period, overall nosocomial infection decreased (prevalence of 16.9% in 1994 to 9.9% in 1998; p=0.04), MRSA transmission rates decreased (2.16 to 0.93 episodes per 10,000 patient-days; p<0.001), and the consumption of alcohol-based handrub solution increased from 3.5 to 15.4 L per 1000 patient-days between 1993 and 1998 (p<0.001).

CONCLUSIONS

The campaign produced a sustained improvement in compliance with hand hygiene, coinciding with a reduction of nosocomial infections and MRSA transmission. The promotion of bedside, antiseptic handrubs largely contributed to the increase in compliance.

Recent work has shown that there may be disadvantages in the use of the chi-square-like goodness-of-fit tests for the logistic regression model proposed by Hosmer and Lemeshow that use fixed groups of the estimated probabilities. A particular concern with these grouping strategies based on estimated probabilities, fitted values, is that groups may contain subjects with widely different values of the covariates. It is possible to demonstrate situations where one set of fixed groups shows the model fits while the test rejects fit using a different set of fixed groups. We compare the performance by simulation of these tests to tests based on smoothed residuals proposed by le Cessie and Van Houwelingen and Royston, a score test for an extended logistic regression model proposed by Stukel, the Pearson chi-square and the unweighted residual sum-of-squares. These simulations demonstrate that all but one of Royston's tests have the correct size. An examination of the performance of the tests when the correct model has a quadratic term but a model containing only the linear term has been fit shows that the Pearson chi-square, the unweighted sum-of-squares, the Hosmer-Lemeshow decile of risk, the smoothed residual sum-of-squares and Stukel's score test, have power exceeding 50 per cent to detect moderate departures from linearity when the sample size is 100 and have power over 90 per cent for these same alternatives for samples of size 500. All tests had no power when the correct model had an interaction between a dichotomous and continuous covariate but only the continuous covariate model was fit. Power to detect an incorrectly specified link was poor for samples of size 100. For samples of size 500 Stukel's score test had the best power but it only exceeded 50 per cent to detect an asymmetric link function. The power of the unweighted sum-of-squares test to detect an incorrectly specified link function was slightly less than Stukel's score test. We illustrate the tests within the context of a model for factors associated with low birth weight.