Determination of pattern centre in EBSD using the moving-screen technique.

The 'moving-screen' or 'pattern magnification' method of calibration for electron backscatter diffraction (EBSD) was reformulated to develop a high-precision technique requiring no crystallographic knowledge of the specimen and no initial estimates of the calibration parameters. The technique depends upon the accurate displacement of the screen and camera assembly. Corresponding points are selected, interactively, from EBSD patterns. It is suggested that, as an alternative, the selection of points from the Hough transform could lead to a completely automated routine.

Exchange dynamics of nitric oxide in the human nose.

Nasal nitric oxide (NO) exchange dynamics are poorly understood but potentially are of importance, inasmuch as they may provide insight into the NO-related physiology of the bronchial tree. In healthy human volunteers, NO output was assessed by isolating the nasal cavity through elevation of the soft palate and application of tight-fitting nasal olives. Mean NO output was 334 nl/min and was a positive function of gas flow. With the use of a mathematical model and the introduction of nonzero concentrations of NO, the diffusing capacity for NO in the nose (DNO) and the mucosal NO concentration (Cw) were determined. DNO ranged from 0.52 to 2.98 x 10(-3) nl x s(-1) x ppb(-1) and Cw from 1,236 to 8,947 ppb. Cw declined with increasing gas flow, while DNO was constant. NO output declined with luminal hypoxia, particularly at oxygen tensions <10%. Measurement of nasal DNO and Cw is easy using this method, and the range of intersubject values of Cw raises the possibility of interindividual differences in NO-dependent nasal physiology.

Point mutations in the murine fumarylacetoacetate hydrolase gene: Animal models for the human genetic disorder hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic failure, whereas a chronic form leads to gradual development of liver disease often accompanied by renal dysfunction, childhood rickets, neurological crisis, and hepatocellular carcinoma. Mice homozygous for certain chromosome 7 deletions of the albino Tyr; c locus that also include Fah die perinatally as a result of liver dysfunction and exhibit a complex syndrome characterized by structural abnormalities and alterations in gene expression in the liver and kidney. Here we report that two independent, postnatally lethal mutations induced by N-ethyl-N-nitrosourea and mapped near Tyr are alleles of Fah. The Fah(6287SB) allele is a missense mutation in exon 6, and Fah(5961SB) is a splice mutation causing loss of exon 7, a subsequent frameshift in the resulting mRNA, and a severe reduction of Fah mRNA levels. Increased levels of the diagnostic metabolite succinylacetone in the urine of the Fah(6287SB) and Fah(5961SB) mutants indicate that these mutations cause a decrease in Fah enzymatic activity. Thus, the neonatal phenotype present in both mutants is due to a deficiency in Fah caused by a point mutation, and we propose Fah(5961SB) and Fah(6287SB) as mouse models for acute and chronic forms of human HT1, respectively.

Effects of ENU dosage on mouse strains.

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F(1) hybrids to ENU. The results show that most F(1) hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.

Benign prognosis of never-symptomatic carotid occlusion.

OBJECTIVE: To determine the prognosis of asymptomatic carotid artery occlusion. BACKGROUND: As opposed to symptomatic carotid occlusion, little information is available on the prognosis of asymptomatic carotid occlusion. METHOD: Thirty never-symptomatic and 81 symptomatic patients with carotid occlusion underwent baseline assessment of 15 risk factors together with PET measurements of oxygen extraction fraction (OEF). Every 6-month telephone contact recorded interval medical treatment and subsequent stroke occurrence during an average follow-up of 32 months. Patients, treating physicians, and an end point adjudicator were blinded to PET results. RESULTS: Ischemic stroke occurred in 1 of 30 of never-symptomatic patients (3.3%) and 15 of 81 of symptomatic patients (18.5%; p = 0.03). No strokes in the carotid territory distal to the occluded vessel occurred in the never-symptomatic patients. Multivariate analysis of baseline risk factors for all 111 patients revealed that age, plasma fibrinogen level, and PET findings of high OEF distal to the occluded carotid artery were the only independent predictors of subsequent stroke (p < 0.05). Previous ipsilateral hemispheric or retinal symptoms was not a significant predictive variable. The lower risk of stroke in never-symptomatic patients was associated with a lower incidence of high OEF (4 of 30) as opposed to symptomatic patients (39 of 81; p = 0.002), but there was no significant difference in age or fibrinogen level. CONCLUSIONS: Never-symptomatic carotid occlusion carries a very low risk of subsequent ischemic stroke. This benign prognosis is associated with a low incidence of cerebral hemodynamic compromise in these patients. These data support further the importance of hemodynamic factors in the pathogenesis of ischemic stroke in patients with carotid occlusion.

Compensatory mechanisms for chronic cerebral hypoperfusion in patients with carotid occlusion.

BACKGROUND AND PURPOSE: The purpose of this experiment was to assess long-term cerebral hemodynamic and metabolic changes in patients with increased oxygen extraction fraction (OEF) in the hemisphere distal to an occluded carotid artery who remain free of stroke. Methods--Ten patients with increased OEF and no interval stroke underwent repeated positron emission tomography examinations 12 to 59 months after the initial examination. Quantitative regional measurements of cerebral blood flow, cerebral blood volume, cerebral rate of oxygen metabolism (CMRO2), and OEF were obtained. Regional measurements of the cerebral rate of glucose metabolism (CMRGlc) were made on follow-up in 5 patients. Statistical significance (P<0.05) was measured with t tests and linear regression analysis. RESULTS: The ipsilateral/contralateral OEF ratio declined from a mean of 1.16 to 1.08 (P=0.022). Greater reductions were seen with longer duration of follow-up (P=0.023, r=0.707). The cerebral blood flow ratio improved from 0.81 to 0.85 (P=0.021). No change in cerebral blood volume or CMRO2 was observed. CMRGlc was reduced in the ipsilateral hemisphere (P=0.001 compared with normal), but the CMRO2/CMRGlc ratio was normal. CONCLUSIONS: Increased OEF improves in patients with carotid occlusion and no interval stroke. This improvement in OEF is due to an improvement in collateral blood flow.

N-ethyl-N-nitrosourea mutagenesis of a 6- to 11-cM subregion of the Fah-Hbb interval of mouse chromosome 7: Completed testing of 4557 gametes and deletion mapping and complementation analysis of 31 mutations.

An interval of mouse chromosome (Chr) 7 surrounding the albino (Tyr; c) locus, and corresponding to a long 6- to 11-cM Tyr deletion, has been the target of a large-scale mutagenesis screen with the chemical supermutagen N-ethyl-N-nitrosourea (ENU). A segment of Chr 7, from a mutagenized genome bred from ENU-treated males, was made hemizygous opposite the long deletion for recognition and recovery of new recessive mutations that map within the albino deletion complex. Over 6000 pedigrees were analyzed, and 4557 of these were completely tested for mutations specifying both lethal and gross visible phenotypes. Thirty-one nonclustered mutations were identified and assigned to 10 complementation groups by pairwise trans-complementation crosses. Deletion-mapping analyses, using the extensive series of radiation-induced Tyr deletions, placed the loci defined by each of these complementation groups into defined intervals of the Tyr-region deletion map, which facilitates the identification of each locus on physical and transcription maps of the region. These mutations identified seven new loci and provided new ENU-induced alleles at three previously defined loci. Interestingly, no mutations were recovered that recapitulated three phenotypes defined by analysis of homozygous or partially complementing albino deletions. On the basis of our experience with this screen, we discuss a number of issues (e.g., locus mutability, failure to saturate, number of gametes to screen, allelic series) of concern when application of chemical mutagenesis screens to megabase regions of the mouse genome is considered.

A fatality due to the intranasal abuse of methylphenidate (Ritalin).

A fatality in a teenager from the recreational intranasal abuse of methylphenidate (Ritalin) is reported. The prescribed use of methylphenidate (Ritalin) in the treatment of attention deficit and/or hyperactivity disorder is widespread. The intranasal abuse of methylphenidate (Ritalin) among teenagers is becoming increasingly more recognized. Previous deaths from the parenteral abuse of methylphenidate (Ritalin) have been reported. This fatality is the first reported from its intranasal abuse.

Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear.

The cochlea and vestibular structures of the inner ear labyrinth develop from the otic capsule via step-wise regional and cell fate specification. Each inner ear structure contains a sensory epithelium, composed of hair cells, the mechanosensory transducers, and supporting cells. We examined the spatio-temporal expression of genes in the Notch signaling pathway, Notch receptors (Notch1-4) and two ligands, Jagged1 and Delta1, in the developing mammalian inner ear. Our results show that Notch1 and Jagged1 are first expressed in the otic vesicle, likely involved in differentiation of the VIIIth nerve ganglion neurons, and subsequently within the inner ear sensory epithelia, temporally coincident with initial hair cell differentiation. Notch1 expression is specific to hair cells and Jagged1 to supporting cells. Their expression persists into adult. Notch2, Notch3, Notch4, and Delta1 are excluded from the inner ear epithelia. These data support the hypothesis that Notch signaling is involved in hair cell differentiation during inner ear morphogenesis.

Importance of hemodynamic factors in the prognosis of symptomatic carotid occlusion.

CONTEXT: The relative importance of hemodynamic factors in the pathogenesis and treatment of stroke in patients with carotid artery occlusion remains controversial. OBJECTIVE: To test the hypothesis that stage II cerebral hemodynamic failure (increased oxygen extraction measured by positron emission tomography [PET]) distal to symptomatic carotid artery occlusion is an independent risk factor for subsequent stroke in medically treated patients. DESIGN AND SETTING: Prospective, blinded, longitudinal cohort study of patients referred from a group of regional hospitals between 1992 and 1996. PATIENTS: From 419 subjects referred, 81 with previous stroke or transient ischemic attack in the territory of an occluded carotid artery were enrolled. All were followed up to completion of the study, with average follow-up of 31.5 months. MAIN OUTCOME MEASURES: Telephone contact every 6 months recorded the subsequent occurrence of all stroke, ipsilateral ischemic stroke, and death. RESULTS: Stroke occurred in 12 of 39 patients with stage II hemodynamic failure and in 3 of 42 patients without (P = .005); stroke was ipsilateral in 11 of 39 patients with stage II hemodynamic failure and in 2 of 42 patients without (P = .004). Six deaths occurred in each group (P = .94). The age-adjusted relative risk conferred by stage II hemodynamic failure was 6.0 (95% confidence interval [CI], 1.7-21.6) for all stroke and 7.3 (95% CI, 1.6-33.4) for ipsilateral stroke. CONCLUSIONS: Stage II hemodynamic failure defines a subgroup of patients with symptomatic carotid occlusion who are at high risk for subsequent stroke when treated medically. A randomized trial evaluating surgical revascularization in this high-risk subgroup is warranted.

Increased oxygen extraction fraction is associated with prior ischemic events in patients with carotid occlusion.

BACKGROUND AND PURPOSE: The purpose of our study was to investigate the relationship between misery perfusion (increased oxygen extraction fraction, OEF) and baseline risk factors in patients with carotid occlusion. METHODS: One-hundred seventeen patients with atherosclerotic carotid occlusion were studied prospectively by clinical evaluation, laboratory testing, and positron emission tomography (PET). PET measurements of cerebral blood flow (CBF), cerebral blood volume (CBV), and OEF were made on enrollment in the study. Increased ipsilateral OEF was identified by comparison with 18 normal control subjects. Twenty-five baseline clinical, epidemiological, and arteriographic risk factors were assessed on study entry. Student t tests, chi(2) tests, and Fisher exact tests with Bonferroni correction were used to assess statistical significance (P<.05). RESULTS: Of 117 patients, 44 had increased OEF distal to the occluded carotid and 73 had normal OEFs. Thirty-nine of the 81 patients with prior ipsilateral ischemic symptoms had high OEFs (42%), whereas only 5 of the 31 asymptomatic patients had high OEFs (16%, P<.001). All of the other baseline risk factors were similar between the two groups of patients. CONCLUSIONS: Investigations of the relationship between hemodynamic factors and stroke risk must take into account the lower frequency of hemodynamic abnormalities in asymptomatic patients.

Hematopoietic deficiencies in c-mpl and TPO knockout mice.

Thrombopoietin (TPO) is the primary regulatory of megakaryocyte (Meg) and platelet production. Its receptor, c-mpl, is a member of the cytokine receptor superfamily. Major insight into the physiological role of this receptor/ligand pair came from the study of mice carrying disrupted alleles of these two genes. Both TPO and c-mpl knockout mice are viable, but have a 90% reduction in platelet counts. Their thrombocytopenia is caused by a reduction in progenitor cell numbers and a decrease in Meg ploidy. However, the Megs and platelets produced in the absence of TPO or c-mpl appear morphologically and functionally normal indicating that, in vivo, the main role of TPO is to control their numbers, rather than their maturation. In addition to its effect on the Meg lineage, TPO also affects hematopoietic stem cells as measured by a reduction of the repopulating capacity of bone marrow cells from c-mpl-deficient mice. Finally, analysis of these gene targeted mice provided substantial evidence to a model where the circulating TPO level is directly regulated by the platelet mass through binding to c-mpl receptors present at the platelet surface. This elegant feedback mechanism allows a tight regulation of the amount of TPO available to stimulate megakaryocytopoiesis.

Mutations in the murine fitness 1 gene result in defective hematopoiesis.

Identification and characterization of mutations that disrupt normal hematopoiesis are essential for understanding the genetic pathways that control the development and regulation of the mammalian hematopoietic system. Previously, the fitness 1 gene was identified by five, independent mutations in N-ethyl-N-nitrosourea (ENU) saturation mutagenesis experiments within the albino (c) region of mouse chromosome 7 (MMU7). We report here that fit1 mutants are anemic, display numerous peripheral blood defects, and are deficient in early hematopoietic progenitor cell populations. The number of both erythroid and myeloid progenitors, as well as B cells, are reduced. These results implicate fit1 involvement in normal hematopoiesis and suggest that further characterization of the fit1 gene, and the five presumed point mutations of the gene, will lead to an improved understanding of normal hematopoiesis in the mouse.

Neurofilament triplet protein interactions: evidence for the preferred formation of NF-L-containing dimers and a putative function for the end domains.

In this report we examine the molecular interactions that lead to formation of neurofilaments, the intermediate filaments in neurons. Using the yeast two-hybrid system, we found that the rod domains of all three NF triplet proteins interacted strongly with one another and with rod domains of the Type III IF proteins, vimentin and desmin. A slight preference toward NF-L-containing dimers was observed over ones not containing NF-L. Interactions among the full length NF triplet proteins exhibited more specificity. Full length NF-L had only a relatively weak interaction with another full length NF-L molecule, but reacted more robustly with full length NF-M or NF-H lacking only part of the head domain. No homologous or heterologous dimerization of NF-M and NF-H was detectable. These results support the hypothesis that neurofilaments are obligate heteropolymers and that heterodimeric subunits are the preferred building blocks. They further suggest that the mechanism that specifies heterodimeric interaction among the NF triplet proteins resides in the end domains.

Pleiotropy in microdeletion syndromes: neurologic and spermatogenic abnormalities in mice homozygous for the p6H deletion are likely due to dysfunction of a single gene.

Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p6H deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p6H deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profound abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p6H deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p6H deletion homozygotes. Because EtNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p6H deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans.

Correction of distortion in US images caused by subcutaneous tissues: results in tissue phantoms and human subjects.

PURPOSE: To correct distortion in ultrasonic images caused by refraction at fat-muscle interfaces in the subcutaneous tissues. MATERIALS AND METHODS: A forward-propagation technique was developed that used a priori information of the acoustic properties of the layers. The thickness and shape of these tissues were measured, and a correction was applied for the different velocities in the tissues. A synthetic-aperture scanning technique was used to allow correction to be applied with data obtained in a single scan. The technique was tested in three tissue phantoms with overlying aberrating layers (six images) and in eight volunteer subjects (37 images). The superior mesenteric artery and the aorta were used as test sites within the body, because it is relatively easy to obtain double images of these vessels owing to refraction of the scanning beam by the rectus muscles. RESULTS: Distortions such as double-image artifacts and texture disruption were corrected with use of this technique. Six of the six phantom images and 22 of the 37 images in humans were corrected with use of this technique. CONCLUSION: The forward-propagation technique compensates for refraction at subcutaneous fat-muscle interfaces.

Genetic and physical mapping of the fitness 1 (fit1) locus within the Fes-Hbb region of mouse chromosome 7.

Mutations at the fit1 locus affect normal pre- and post-natal development by retarding growth and reducing viability. We report mapping of the fit1 locus, by trans-complementation crosses to mice carrying deletions of the albino (c) locus in Chromosome (Chr) 7, to a subregion of the c-deletion complex within the Mod2-sh1 interval. The fit1 locus, which is currently defined by five N-ethyl-N-nitrosourea (ENU)-induced mutations, was found to map in a subregion between the eed and exed loci. A restriction fragment containing a deletion breakpoint that genetically defines the proximal border of fit1 was cloned, providing a DNA probe (RN302) that maps proximal to fit1. Long-range mapping with this probe, and with a DNA probe that maps distal to the fit1 interval, established that the region containing at least part of the fit1 gene is 530 kb or less. Positioning of fit1 between deletion breakpoints, and the isolation and mapping of a DNA probe proximal to it, should facilitate the cloning and molecular characterization of fit1, as well as of the eed locus and the tightly linked l(7)5Rn and l(7)6Rn loci.

Deletion mapping of four loci defined by N-ethyl-N-nitrosourea-induced postimplantation-lethal mutations within the pid-Hbb region of mouse chromosome 7.

As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletion map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethals was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5).

N-ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse chromosome 7.

Two loci [l(7)5Rn and l(7)6Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the albino (c) locus in Chromosome (Chr) 7. Both loci were found to map to the subregion of the Mod-2-sh-1 interval that contains the eed (embryonic ectoderm development) locus, eed has been defined by the inability of embryos homozygous for certain c deletions to develop beyond the early stages of gastrulation. Evidence for at least two loci necessary for normal prenatal development, rather than one locus, that map within the eed interval came from the observation that two prenatally lethal mutations, 3354SB [l(7)5Rn3354SB] and 4234SB [l(7)6Rn4234SB], could complement each other in trans, but could not each be complemented individually by c deletions known to include the eed locus. A somewhat leaky allele of l(7)5Rn [l(7)5Rn1989SB] was also recovered, in which hemizygotes are often stillborn and homozygotes exhibit variable fitness and survival. The mapping of the loci defined by these mutations is likely to be useful for genetic, molecular, and phenotypic characterization of the eed region, and mutations at either locus (or both loci) may contribute to the eed phenotype.