Mechanism of hyperphagia contributing to obesity in brain-derived neurotrophic factor knockout mice.

Global-heterozygous and brain-specific homozygous knockouts (KOs) of brain-derived neurotrophic factor (BDNF) cause late- and early-onset obesity, respectively, both involving hyperphagia. Little is known about the mechanism underlying this hyperphagia or whether BDNF loss from peripheral tissues could contribute to overeating. Since global-homozygous BDNF-KO is perinatal lethal, a BDNF-KO that spared sufficient brainstem BDNF to support normal health was utilized to begin to address these issues. Meal pattern and microstructure analyses suggested overeating of BDNF-KO mice was mediated by deficits in both satiation and satiety that resulted in increased meal size and frequency and implicated a reduction of vagal signaling from the gut to the brain. Meal-induced c-Fos activation in the nucleus of the solitary tract, a more direct measure of vagal afferent signaling, however, was not decreased in BDNF-KO mice, and thus was not consistent with a vagal afferent role. Interestingly though, meal-induced c-Fos activation was increased in the dorsal motor nucleus of the vagus nerve (DMV) of BDNF-KO mice. This could imply that augmentation of vago-vagal digestive reflexes occurred (e.g., accommodation), which would support increased meal size and possibly increased meal number by reducing the increase in intragastric pressure produced by a given amount of ingesta. Additionally, vagal sensory neuron number in BDNF-KO mice was altered in a manner consistent with the increased meal-induced activation of the DMV. These results suggest reduced BDNF causes satiety and satiation deficits that support hyperphagia, possibly involving augmentation of vago-vagal reflexes mediated by central pathways or vagal afferents regulated by BDNF levels.

Development of the vagal innervation of the gut: steering the wandering nerve.

BACKGROUND: The vagus nerve is the major neural connection between the gastrointestinal tract and the central nervous system. During fetal development, axons from the cell bodies of the nodose ganglia and the dorsal motor nucleus grow into the gut to find their enteric targets, providing the vagal sensory and motor innervations respectively. Vagal sensory and motor axons innervate selective targets, suggesting a role for guidance cues in the establishment of the normal pattern of enteric vagal innervation. PURPOSE: This review explores known molecular mechanisms that guide vagal innervation in the gastrointestinal tract. Guidance and growth factors, such as netrin-1 and its receptor, deleted in colorectal cancer, extracellular matrix molecules, such as laminin-111, and members of the neurotrophin family of molecules, such as brain-derived neurotrophic factor have been identified as mediating the guidance of vagal axons to the fetal mouse gut. In addition to increasing our understanding of the development of enteric innervation, studies of vagal development may also reveal clinically relevant insights into the underlying mechanisms of vago-vagal communication with the gastrointestinal tract.

Ultrastructural evidence for communication between intramuscular vagal mechanoreceptors and interstitial cells of Cajal in the rat fundus.

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.

Activating mutations in NOTCH1 in acute myeloid leukemia and lineage switch leukemias.

Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.

Homosexual behavior in wild Sumatran orangutans (Pongo pygmaeus abelii).

Wild male Sumatran orangutans at two study sites engaged in homosexual behavior. These observations demonstrate that homosexual behavior is not an artifact of captivity or contact with humans. In separate instances, homosexual behavior was associated with affiliative and agonistic behaviors. These observations add orangutans to the list of primates in which homosexual behavior forms part of the natural repertoire of sexual or sociosexual behavior.

Functional communication between endogenous BRCA1 and its partner, BARD1, during Xenopus laevis development.

The breast and ovarian susceptibility protein 1 (BRCA1) heterodimerizes with its structural relative, the BRCA1-associated RING domain protein (BARD1), which may have tumor suppressing function in its own right. Both proteins have evolved from a common evolutionary ancestor, and both exist in Xenopus laevis where, similar to their mammalian homologs, they form functional heterodimers. Depleting frog embryos of either BARD1 or BRCA1 led to similar and widely defective developmental phenotypes as well as depletion of the other polypeptide due to its decreased stability. Thus, each protein, in part, controls the abundance, stability, and function of the other, and these effects are heterodimerization-dependent. The interdependent nature of BRCA1 and BARD1 function supports the view that BARD1/BRCA1 heterodimers play a major role in breast and ovarian cancer suppression.

Neurotrophin-4 deficient mice have a loss of vagal intraganglionic mechanoreceptors from the small intestine and a disruption of short-term satiety.

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs) are the two putative mechanoreceptors that the vagus nerve supplies to gastrointestinal smooth muscle. To examine whether neurotrophin-4 (NT-4)-deficient mice, which have only 45% of the normal number of nodose ganglion neurons, exhibit selective losses of these endings and potentially provide a model for assessing their functional roles, we inventoried IGLEs and IMAs in the gut wall. Vagal afferents were labeled by nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase, and a standardized sampling protocol was used to map the terminals in the stomach, duodenum, and ileum. NT-4 mutants had a substantial organ-specific reduction of IGLEs; whereas the morphologies and densities of both IGLEs and IMAs in the stomach were similar to wild-type patterns, IGLEs were largely absent in the small intestine (90 and 81% losses in duodenum and ileum, respectively). Meal pattern analyses revealed that NT-4 mutants had increased meal durations with solid food and increased meal sizes with liquid food. However, daily total food intake and body weight remained normal because of compensatory changes in other meal parameters. These findings indicate that NT-4 knock-out mice have a selective vagal afferent loss and suggest that intestinal IGLEs (1) may participate in short-term satiety, probably by conveying feedback about intestinal distension or transit to the brain, (2) are not essential for long-term control of feeding and body weight, and (3) play different roles in regulation of solid and liquid diet intake.

Analysis of the ethylene response in the epinastic mutant of tomato.

Ethylene can alter plant morphology due to its effect on cell expansion. The most widely documented example of ethylene-mediated cell expansion is promotion of the "triple response" of seedlings grown in the dark in ethylene. Roots and hypocotyls become shorter and thickened compared with controls due to a reorientation of cell expansion, and curvature of the apical hook is more pronounced. The epinastic (epi) mutant of tomato (Lycopersicon esculentum) has a dark-grown seedling phenotype similar to the triple response even in the absence of ethylene. In addition, in adult plants both the leaves and the petioles display epinastic curvature and there is constitutive expression of an ethylene-inducible chitinase gene. However, petal senescence and abscission and fruit ripening are all normal in epi. A double mutant (epi/epi;Nr/Nr) homozygous for both the recessive epi and dominant ethylene-insensitive Never-ripe loci has the same dark-grown seedling and vegetative phenotypes as epi but possesses the senescence and ripening characteristics of Never-ripe. These data suggest that a subset of ethylene responses controlling vegetative growth and development may be constitutively activated in epi. In addition, the epi locus has been placed on the tomato RFLP map on the long arm of chromosome 4 and does not demonstrate linkage to reported tomato CTR1 homologs.

C-Kit mutant mice have a selective loss of vagal intramuscular mechanoreceptors in the forestomach.

Intramuscular arrays are one of two major classes of vagal afferent mechanoreceptors that innervate the smooth muscle wall of the proximal gastrointestinal tract. They consist of rectilinear telodendria that distribute in the muscle sheets, parallel to the long axes of muscle fibers. Intramuscular arrays appear to make direct contact with the muscle fibers, but they also course on, and form appositions with, intramuscular interstitial cells of Cajal. These complexes formed by intramuscular arrays and intramuscular interstitial cells of Cajal suggest that intramuscular arrays might require either structural or trophic support of the interstitial cells of Cajal for normal differentiation and/or maintenance. To evaluate this hypothesis, we have examined the morphology and distribution of vagal afferent endings in the c-Kit mutant mouse that lacks intramuscular interstitial cells of Cajal. Vagal afferents were labeled by nodose ganglion injection of either wheat germ agglutinin-horseradish peroxidase conjugate or a tagged dextran, and the labeled afferent terminals in the stomach were mapped using a standardized quantitative sampling scheme. Intramuscular arrays were dramatically reduced (in circular muscle by 63%; in longitudinal muscle by 78%) in the c-Kit mutant mice relative to their wild-type littermates. Additionally, a substantial number of the surviving axons and terminals in the mutant stomachs were morphologically aberrant. Moreover, the loss of intramuscular arrays in mutants appeared to be selective: the structure, distribution and density of intraganglionic laminar endings, i.e., the other vagal mechanoreceptors in smooth muscle, were not significantly altered. Finally, the conspicuous decrease in intramuscular array density in mutants was associated with a non-significant trend toward loss of nodose ganglion neurons. Collectively these findings suggest that interstitial cells are required for the normal development or maintenance of vagal intramuscular arrays. Therefore, the c-Kit mutant mouse will be valuable for determining the role(s) of interstitial cells in intramuscular array development as well as for providing an animal model with the intramuscular array class of vagal afferents selectively ablated.

Preparation of DNA from fixed, paraffin-embedded tissue.

Purification of DNA from fixed, paraffin-embedded tissue (PET) requires the removal of paraffin, proteins, and, in some cases, heavy metal ions. The resulting DNA is not generally suitable for Southern blot analysis. However, the polymerase chain reaction (PCR) can be used to amplify short regions of this DNA. The Basic Protocol is rapid and involves only a small number of manipulations, thus minimizing the possibility of contamination. The Alternate Protocol involves additional steps including extractions with xylenes and phenol/chloroform. This procedure is recommended as the primary approach when tissue has not been fixed in an optimal manner or as a secondary approach when the Basic Protocol has failed to produce satisfactory DNA.

Vagal afferent innervation of smooth muscle in the stomach and duodenum of the mouse: morphology and topography.

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs), the two putative mechanoreceptors that the vagus nerve supplies to the gastrointestinal smooth muscle, have been characterized almost exclusively in the rat. To provide normative inventories of these afferents for the mouse, the authors examined the endings in the stomach and small intestine of three strains used as backgrounds for gene manipulations (i.e., C57, 129/SvJ, and WBB6). Animals received nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase or dextran-tetramethylrhodamine conjugated to biotin. The horseradish peroxidase tissue was processed with tetramethylbenzidine and was used to map the distributions and densities of the two endings; the dextran material was counterstained with c-Kit immunohistochemistry to assess interactions between intramuscular arrays and interstitial cells of Cajal. IGLEs and IMAs constituted the vagal innervation of mouse gastric and duodenal smooth muscle. IGLE morphology and distributions, with peak densities in the corpus-antrum, were similar in the three strains of mice and comparable to those observed in rats. IMAs varied in complexity from region to region but tended to be simpler (fewer telodendria) in mice than in rats. IMAs were most concentrated in the forestomach and sphincters in mice, as in rats, but the topographic distributions of the endings varied both between strains of mice (subtly) and between species (more dramatically). IMAs appeared to make appositions with both interstitial cells and smooth muscle fibers. This survey should make it practical to assay the effects of genetic (e.g., knockout) and experimental (e.g., regeneration) manipulations affecting visceral afferents and their target tissues.

Characterization of hereditary nonpolyposis colorectal cancer families from a population-based series of cases.

BACKGROUND: The incidence of hereditary nonpolyposis colon cancer (HNPCC) in the general population is not well defined because of the lack of large population-based studies. We characterized the incidence of HNPCC in a large, population-based cohort of colorectal cancer probands and analyzed the location of colorectal tumors. METHODS: Of the participating 1134 probands from three counties in Southern California, 907 had a negative family history of colorectal cancer and 227 had a positive family history of colorectal cancer. In addition, 11 referral case subjects with HNPCC were used to study mutation frequencies in two mismatch repair genes (MSH2 and MLH1) and microsatellite instability. All statistical tests were two-sided. RESULTS: Among the probands diagnosed in Orange County during 1994 (population-based sample, all ages), five were consistent with the Amsterdam criteria for HNPCC (0.9%; 95% confidence interval [CI] = 0. 3%-2.1%). Among probands diagnosed at less than 65 years of age-from the wider three-county area and a longer time span-16 (2.1%; 95% CI = 1.2%-3.4%) had a clinical history consistent with the Amsterdam criteria for HNPCC. Five (approximately 45%) of 11 of the referral HNPCC case subjects had a mutation in MSH2 or MLH1 and also showed microsatellite instability. The family members of case subjects with mutations tended to show an earlier age at diagnosis of HNPCC and more multiple primary cancers than those of case subjects without detectable mutations. Many of the known characteristics of HNPCC, including the presence of ureteral and endometrial cancers, were seen in both sets of families. The previously reported proximal location of colorectal tumors in HNPCC kindreds was not seen in the population-based dataset but was similar to the location reported in the referral cases. CONCLUSIONS: On the basis of our data, we believe that the prevalence of HNPCC in the general population is likely to be closer to 1% than to 5%. Furthermore, our study suggests that some previously reported characteristics of HNPCC, such as the proximal location of tumors in the syndrome, may not always hold true in a population-based sample.

Sensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations in MSH2 and MLH1.

BACKGROUND AND AIMS: There are multiple criteria for the clinical diagnosis of hereditary non-polyposis colorectal cancer (HNPCC). The value of several of the newer proposed diagnostic criteria in identifying subjects with mutations in HNPCC associated mismatch repair genes has not been evaluated, and the performance of the different criteria have not been formally compared with one another. METHODS: We classified 70 families with suspected hereditary colorectal cancer (excluding familial adenomatous polyposis) by several existing clinical criteria for HNPCC, including the Amsterdam criteria, the Modified Amsterdam criteria, the Amsterdam II criteria, and the Bethesda criteria. The results of analysis of the mismatch repair genes MSH2 and MLH1 by full gene sequencing were available for a proband with colorectal neoplasia in each family. The sensitivity and specificity of each of the clinical criteria for the presence of MSH2 and MLH1 mutations were calculated. RESULTS: Of the 70 families, 28 families fulfilled the Amsterdam criteria, 39 fulfilled the Modified Amsterdam Criteria, 34 fulfilled the Amsterdam II criteria, and 56 fulfilled at least one of the seven Bethesda Guidelines for the identification of HNPCC patients. The sensitivity and specificity of the Amsterdam criteria were 61% (95% CI 43-79) and 67% (95% CI 50-85). The sensitivity of the Modified Amsterdam and Amsterdam II criteria were 72% (95% CI 58-86) and 78% (95% CI 64-92), respectively. Overall, the most sensitive criteria for identifying families with pathogenic mutations were the Bethesda criteria, with a sensitivity of 94% (95% CI 88-100); the specificity of these criteria was 25% (95% CI 14-36). Use of the first three criteria of the Bethesda guidelines only was associated with a sensitivity of 94% and a specificity of 49% (95% CI 34-64). CONCLUSIONS: The Amsterdam criteria for HNPCC are neither sufficiently sensitive nor specific for use as a sole criterion for determining which families should undergo testing for MSH2 and MLH1 mutations. The Modified Amsterdam and the Amsterdam II criteria increase sensitivity, but still miss many families with mutations. The most sensitive clinical criteria for identifying subjects with pathogenic MSH2 and MLH1 mutations were the Bethesda Guidelines; a streamlined version of the Bethesda Guidelines may be more specific and easier to use in clinical practice.

Highly selective isolation of unknown mutations in diverse DNA fragments: toward new multiplex screening in cancer.

Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (T-->G) and wild-type DNA-fragment populations. Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY. Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch. For PCR amplification, synthetic linkers are then ligated to the DNA fragments. Biotinylated DNA is then isolated and PCR amplified. Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation. This method correctly detects a single T-->G transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene. In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified. The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude. Homozygous and heterozygous p53 regions (G-->T, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated. This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest.

The previously identified r3/r5 repressor may require the cooperation of additional negative elements for rhombomere restriction of Hoxb1.

Hoxb1 has several rhombomere-specific roles in hindbrain development and may contribute to development of a small number of tissues outside the hindbrain. To gain insight into the regulation of late Hoxb1 expression in these structures, several regions of the Hoxb1 locus were systematically evaluated for their ability to control late Hoxb1 expression in transgenic mouse embryos. This was achieved by progressive enlargement of the portion of the Hoxb1 locus used to control reporter gene expression. Unexpectedly, the previously identified rhombomere 4 (r4) enhancer and r3/r5 repressor that were thought to be sufficient for r4 restriction of Hoxb1 produced continuous expression throughout much of the length of the central nervous system (CNS) and in several structures outside the nervous system. However, adjacent regions of the Hoxb1 locus, in combination with the r4 enhancer and r3/r5 repressor were capable of restricting most expression to r4 in the hindbrain, and to gut epithelium/mesoderm, caudal spinal cord and caudal paraxial mesoderm outside the hindbrain. Expression that occurred outside r4 in the brains of the majority of founder embryos was confined to scattered cells in specific regions. These cells may have arisen in r4 and then migrated into adjacent brain regions as rhombomere lineage restrictions dissipated. Alternatively, these dispersed stained cells may have originated outside r4 implying that Hoxb1 cannot be accurately regulated in a consistent manner outside the Hox complex.

Germ-line msh6 mutations in colorectal cancer families.

Hereditary nonpolyposis colorectal carcinoma (HNPCC) is due primarily to inherited mutations in two mismatch repair genes, MSH2 and MLH1, whereas germ-line mutations in other mismatch repair genes are rare. We examined the frequency of germ-line msh6 mutations in a population-based series of 140 colorectal cancer patients, including 45 sporadic cases, 91 familial non-HNPCC cases, and 4 HNPCC cases. Among the 91 population-based familial non-HNPCC cases, germ-line msh6 mutations were found in 6 patients (7.1% of probands analyzed; median age at diagnosis, 61 years). These mutations included a splice site mutation, a frameshift mutation, two missense mutations that were demonstrated to be loss of function mutations, and two missense mutations for which functional studies were not possible. In contrast, germ-line msh6 mutations were not found in any of the 45 sporadic cases and the 4 HNPCC cases in the population-based series or in the second series of 58 clinic-based, primarily HNPCC families. Our data suggest that germ-line msh6 mutations predispose individuals to primarily late-onset, familial colorectal carcinomas that do not fulfill classic criteria for HNPCC.

Interpretation of genetic test results for hereditary nonpolyposis colorectal cancer: implications for clinical predisposition testing.

CONTEXT: Genetic testing for cancer predisposition is evolving from purely research applications to affecting clinical management. OBJECTIVE: To determine how often genetic test results for hereditary nonpolyposis colorectal cancer (HNPCC) can be definitively interpreted and used to guide clinical management. DESIGN: Case-series study conducted in 1996 to 1998 in which a complete sequence analysis of hMSH2 and hMLH1 coding sequence and flanking intronic regions was performed. Mutations were categorized as protein truncating and missense. In the case of missense alterations, additional analyses were performed in an effort to assess pathogenicity. SETTING AND PARTICIPANTS: Families were identified by self-referral or health care provider referral to a cancer genetics program. Participants and kindreds were classified into 1 of 4 categories: (1) Amsterdam criteria for HNPCC, (2) modified Amsterdam criteria for HNPCC, (3) young age at onset, or (4) HNPCC-variant. In addition, each proband was classified according to the Bethesda guidelines for identification of individuals with HNPCC. MAIN OUTCOME MEASURE: Alterations of hMSH2 and hMLH1 genes. RESULTS: Twenty-seven alterations of hMSH2 and hMLH1 were found in 24 of 70 families (34.3%). Of these, deleterious mutations that could be used with confidence in clinical management were identified in 25.7% (18/70) of families. The rates of definitive results for families fulfilling Amsterdam criteria, modified Amsterdam criteria, young age at onset, HNPCC-variant, and Bethesda guidelines were 27 (39.3%), 13 (18.2%), 12 (16.7%), 11 (15.8%), and 21 (30.4%), respectively. The prevalence of missense mutations, genetic heterogeneity of the syndrome, and limited availability of validated functional assays present a challenge in the interpretation of genetic test results of HNPCC families. CONCLUSIONS: The identification of pathogenic mutations in a significant subset of families for whom the results may have marked clinical importance makes genetic testing an important option for HNPCC and HNPCC-like kindreds. However, for the majority of individuals in whom sequence analysis of hMSH2 and hMLH1 does not give a definitive result, intensive follow-up is still warranted.

Comparison of functional testing for resistance to activated protein C and molecular biological testing for factor V R506Q in 370 patients.

OBJECTIVE: To compare functional and molecular biological tests for resistance to activated protein C (APC)/factor V R506Q, the most common cause of familial thrombosis. METHODS: We developed functional and molecular biological tests for resistance to APC/factor V R506Q at our institution and correlated the results for 370 patients studied by both methods. The functional method is based on addition of exogenous APC to an activated partial thromboplastin time-based assay. The molecular biological method is based on polymerase chain reaction followed by endonuclease digestion. RESULTS: Considering the molecular biological test as definitive for detecting the factor V R506Q mutation, the sensitivity of the functional assay was 100%, and the specificity was 74%. The prevalence of the factor V mutation in the population studied was 12% (41 heterozygotes, two homozygotes), and the positive predictive value of the functional assay was 34%. Although a normalized sensitivity ratio (nAPC-SR) less than 0.84 is considered evidence of resistance to APC by functional testing, we found that all patients with factor V R506Q had an nAPC-SR less than or equal to 0.71. When this alternative positive cutoff was used, the specificity of the functional test for factor V R506Q increased to 87%, and the positive predictive value increased to 52%, which constituted a significant improvement. We compared clinical findings from patients with resistance to APC with or without the presence of factor V R506Q, and found that as a group, those with factor V R506Q had a higher incidence of hypercoagulability, but fewer additional risk factors for hypercoagulability. The mechanism of resistance to APC in factor V R506Q-negative individuals is unclear, but may be related to other risk factors for hypercoagulability. CONCLUSIONS: The functional assay for resistance to APC is an excellent screening test for factor V R506Q, but confirmatory molecular biological testing is necessary when the functional test is positive, because of the high false-positive rate.

Accurate, high-throughput "snapshot" detection of hMLH1 mutations by two-dimensional DNA electrophoresis.

The human genome and related projects have resulted in the isolation of a rapidly growing number of genes that cause susceptibility to human cancer. With rare exception, these genes are large and have disease-associated mutations scattered along the length of the genes. Therefore, the development of accurate and cost-efficient mutation detection tests that can scan entire genes singly or in combination is warranted. hMLH1, encoding a mismatch repair enzyme, is a susceptibility gene for hereditary nonpolyposis colon cancer syndrome. This gene comprises 19 exons; mutations are scattered, typical of many susceptibility genes. Here, we present a strategy that combines extensive PCR multiplexing and two-dimensional DNA electrophoresis (Two-Dimensional Gene Scanning, TDGS) to scan accurately for mutations that lie within the exons and splice junctions of hMLH1. All target fragments, designed to have optimal melting characteristics, were prepared in a two-stage PCR--a four-plex long-distance PCR followed by short PCR in two multiplex groups of 10 and 11 amplicons. The mixture of amplicons was subjected to two-dimensional electrophoresis: separation by size in the first dimension and by melting characteristics in the second. Using this design, 41 samples containing known hMLH1 sequence variants or no alterations were blindly subjected to TDGS. All mutations were detected; there were no genuine false-positive or false-negative results. These results confirm that TDGS is a generally applicable, rapid, accurate, and reproducible mutation detection technology that would serve large-scale molecular epidemiologic studies as well as clinical molecular diagnostic purposes.

Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice.

This study identified the organ and cellular distribution of cationic liposome-DNA complexes injected intravenously into CD-1 mice for gene delivery. DOTIM-cholesterol liposomes were labeled with the fluorescent dye CM-Dil and complexed with plasmid DNA encoding the chloramphenicol acetyltransferase reporter gene. The distribution of the complexes was examined in 29 organs and tissues by fluorescence, confocal, and electron microscopy from 5 min to 24 h after injection. The complexes formed clusters in blood, which were cleared within 20 min. Complexes visible by fluorescence microscopy were taken up by endothelial cells, leukocytes, and macrophages and did not leave the vasculature except in the spleen. At 5 min, the complexes formed a patchy coating on the endothelial surface, but by 4 h, they were internalized into endosomes and lysosomes in organ- and vessel-specific patterns. Uptake by capillary endothelial cells was greatest in the lung, ovary, and anterior pituitary, less in muscle and the heart, and nearly absent in the brain and pancreatic islets. In lymph nodes and intestinal Peyer's patches, the uptake was sparse in capillaries but abundant in high endothelial venules. In the liver and spleen, most of the uptake was in Kupffer cells and macrophages. Measurements of chloramphenicol acetyltransferase reporter gene expression were generally consistent with the pattern of uptake by endothelial cells. The uptake and gene expression were accompanied by a decrease in circulating leukocytes and platelets. Overall, our results showed that the complexes were internalized by endothelial cells in organ- and vessel-specific patterns that did not match any previously identified properties of the microvasculature. The unusual distribution of endothelial cell uptake may be explained by a heterogeneously distributed membrane receptor for which the complexes are ligands.