Laforin preferentially binds the neurotoxic starch-like polyglucosans, which form in its absence in progressive myoclonus epilepsy

Lafora disease (LD) is a fatal and the most common form of adolescent-onset progressive epilepsy. Fulminant endoplasmic reticulum (ER)-associated depositions of starch-like long-stranded, poorly branched glycogen molecules [known as polyglucosans, which accumulate to form Lafora bodies (LBs)] are seen in neuronal perikarya and dendrites, liver, skeletal muscle and heart. The disease is caused by loss of function of the laforin dual-specificity phosphatase or the malin E3 ubiquitin ligase. Towards understanding the pathogenesis of polyglucosans in LD, we generated a transgenic mouse overexpressing inactivated laforin to trap normal laforin's unknown substrate. The trap was successful and LBs formed in liver, muscle, neuronal perikarya and dendrites. Using immunogold electron microscopy, we show that laforin is found in close proximity to the ER surrounding the polyglucosan accumulations. In neurons, it compartmentalizes to perikaryon and dendrites and not to axons. Importantly, it binds polyglucosans, establishing for the first time a direct association between the disease-defining storage product and disease protein. It preferentially binds polyglucosans over glycogen in vivo and starch over glycogen in vitro, suggesting that laforin's role begins after the appearance of polyglucosans and that the laforin pathway is involved in monitoring for and then preventing the formation of polyglucosans. In addition, we show that the laforin interacting protein, EPM2AIP1, also localizes on the polyglucosan masses, and we confirm laforin's intense binding to LBs in human LD biopsy material.     

Congenital mesoblastic nephroma: possible prognostic and management value of assessing DNA content.

The case records and pathology of all children with kidney tumours treated in the West Midlands Health Authority Region (WMHAR) from 1957 to 1986 were reviewed. The histology was reviewed by a panel of three paediatric pathologists. Thirteen (6%) out of 211 cases were considered to have congenital mesoblastic nephroma (CMN). Nine were of the conventional type, three of the atypical cellular type, and one mixed. DNA ploidy was investigated and showed two of the tumours to be aneuploid and nine diploid (tissue was not available in the two other cases). The two aneuploid tumours were of atypical cellular and mixed histology, respectively; the diploid tumours were of the conventional type in eight cases and atypical cellular in one. The atypical cellular type has been reported to behave more aggressively, but the benefit of additional treatment after surgery to prevent recurrence remains unclear. Measurement of DNA content by flow cytometry, together with histological subclassification, may be useful in selecting patients who will benefit from further treatment after surgery.     

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.      

Genetic and immunohistochemical analysis of pancreatic acinar cell carcinoma: frequent allelic loss on chromosome 11p and alterations in the APC/beta-catenin pathway

Acinar cell carcinomas (ACCs) are rare malignant tumors of the exocrine pancreas. The specific molecular alterations that characterize ACCs have not yet been elucidated. ACCs are morphologically and genetically distinct from the more common pancreatic ductal adenocarcinomas. Instead, the morphological, immunohistochemical, and clinical features of ACCs overlap with those of another rare pancreatic neoplasm, pancreatoblastoma. We have recently demonstrated a high frequency of allelic loss on chromosome arm 11p and mutations in the APC/beta-catenin pathway in pancreatoblastomas, suggesting that similar alterations might also play a role in the pathogenesis of some ACCs. We analyzed a series of 21 ACCs for somatic alterations in the APC/beta-catenin pathway and for allelic loss on chromosome 11p. In addition, we evaluated the ACCs for alterations in p53 and Dpc4 expression using immunohistochemistry, and for microsatellite instability (MSI) using polymerase chain amplification of a panel of microsatellite markers. Allelic loss on chromosome 11p was the most common genetic alteration in ACCs, present in 50% (6 of 12 informative cases). Molecular alterations in the APC/beta-catenin pathway were detected in 23.5% (4 of 17) of the carcinomas, including one ACC with an activating mutation of the beta-catenin oncogene and three ACCs with truncating APC mutations. One ACC (1 of 13, 7.6%) showed allelic shifts in four of the five markers tested (MSI-high), two (15.4%) showed an allelic shift in only one of the five markers tested (MSI-low), and no shifts were detected in the remaining 10 cases. The MSI-high ACC showed medullary histological features. In contrast, no loss of Dpc4 protein expression or p53 accumulation was detected. These results indicate that ACCs are genetically distinct from pancreatic ductal adenocarcinomas, but some cases contain genetic alterations common to histologically similar pancreatoblastomas.     

Chronic lymphocytic leukemia/small lymphocytic lymphoma associated with IgM paraprotein

We studied the clinicopathologic, immunophenotypic, and cytogenetic features of 26 patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) associated with serum IgM paraprotein. The study group (16 men; 10 women; median age, 64 years; range, 40-82 years) represents approximately 2.5% of CLL/SLL cases at our institution. The paraprotein level ranged from 1 to 14 g/L (median, 4 g/L). Neoplasms in bone marrow were composed of small round lymphocytes arranged in nodular (n = 6), diffuse (n = 5), interstitial (n = 5), or mixed (n = 10) patterns. All cases were positive for monotypic surface immunoglobulin light chain, IgM/IgD, CD5, CD19, CD20, and CD23. CD11c (14/20 [70%]), CD79b (11/19 [58%]), FMC-7 (11/26 [42%]), CD22 (8/20 [40%]), and ZAP-70 (6/19 [32%]) were expressed in subsets of cases. Of 17 bone marrow specimens assessed by conventional cytogenetics, 6 were abnormal and 11 were diploid. The overall survival of this group (median follow-up, 24 months) was not significantly different from that for an age-, sex-and stage-matched group of 52 CLL/SLL patients without IgM paraprotein (P = .60). We conclude that CLL/SLL cases with serum IgM paraprotein are similar to other CLL/SLL cases in their clinicopathologic and immunophenotypic features.     

Timing and expression pattern of carbonic anhydrase II in pancreas.

In the search for genetic markers for assessing the role of duct cells in pancreas growth, we examined whether carbonic anhydrase II (CAII) has ductal cell specificity. We determined the distribution and timing of CAII expression in mouse pancreas from embryonic stage to adult. The pancreatic ducts only start expressing CAII at embryonic day (E) 18.5, with increases after birth. Around E15.5, glucagon-positive cells, but not insulin-positive cells, also express CAII, with further increases by adult. CAII expression was restricted to cells within ductal structures and glucagon-positive cells with no colocalization with any insulin-positive cells at any time. In the human pancreas, CAII expression is restricted to the ducts. Furthermore, the activity of a 1.6-kb fragment of the human promoter with Luciferase assays was moderately strong compared with the cytomegalovirus promoter in human pancreatic duct cell line (PANC-1). Thus, we believe that the CAII gene could serve as a useful pancreatic duct cell marker.     

Generation, annotation, evolutionary analysis, and database integration of 20,000 unique sea urchin EST clusters

Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies, the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analyzed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalization by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalog comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the base of deuterostome evolution. Comparison of the number of gene copies in sea urchins to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. All cDNA clones and filters of all analyzed libraries are available from the resource center of the German genome project at http://www.rzpd.de.     

Human Papillomavirus-Specific Antibody Status in Oral Fluids Modestly Reflects Serum Status in Human Immunodeficiency Virus-Positive Individuals

Serological assays are valuable tools for studies of the epidemiology of human papillomaviruses (HPVs). The efficacy of a less invasive oral-fluid assay for detection of HPV antibodies was examined. Matched serum, saliva, and oral mucosal transudate (OMT) specimens collected from 150 human immunodeficiency virus-seropositive patients were tested for immunoglobulin G antibodies against HPV-6 and HPV-11 combined (HPV-6/11) and HPV-16 capsids. Antibodies to HPV were detected in both types of oral specimens. Seroprevalence rates were 55% for HPV-6/11 and 37% for HPV-16, whereas oral prevalence rates were significantly lower (for HPV-6/11 in saliva, 31%, and in OMT, 19%; for HPV-16 in saliva, 19%, and in OMT, 17%). HPV antibody detection in OMT more accurately reflected the presence of antibodies in serum than did HPV antibody detection in saliva. More stringent saliva assay cutpoints yielded stronger associations between oropositivity and seropositivity; less stringent OMT cutpoints yielded stronger associations between oropositivity and seropositivity. Although HPV antibodies were detected in oral fluids, further optimization of the assay is necessary before oral-fluid testing can be implemented as a reliable alternative to serum testing for HPV.     

Active maintenance of sentence meaning in working memory: evidence from EEG coherences.

The active maintenance of sentence meaning in working memory was investigated using event-related electroencephalogram (EEG) coherences. Participants read a sentence, retained it for 2.5 s, and then verified a statement about its meaning. The sentences contained either three semantically related nouns or unrelated nouns and started either with a what phrase (WH sentences) or not (non-WH sentences), imposing either a high or low demand on verbal working memory. Comprehension accuracy showed an interaction of semantic relatedness and sentence type due to the presence of a relatedness effect (lower accuracy in the unrelated condition) in WH sentences but not in non-WH sentences. During the post-sentence retention interval, EEG coherences also displayed this interaction of relatedness and sentence type. A semantic relatedness effect was obtained in the WH sentences (high demand) but not in the non-WH sentences (low demand). In addition, compared to a pre-sentence baseline and sentence presentation, coherences increased in the 10-14 Hz band during retention and decreased in the 4-6 Hz band. These coherence changes spanned prefrontal and posterior brain regions, possibly reflecting increased synchronization in projection loops between attention control systems in prefrontal cortex and activated meaning representations in semantic memory in posterior cortex. These findings suggest that short-term retention of the meaning of a sentence involves active maintenance in a capacity-limited working memory, accompanied by a heightened inner direction of attention after sentence presentation.     

Complement studies in membranoproliferative glomerulonephritis

Detailed studies of the complement system were carried out in fifteen patients with membranoproliferative glomerulonephritis. The findings of reduced levels of C3 and C7 and of circulating breakdown products of C3 in fresh plasma suggested in vivo complement activation. Low C3 levels were associated with the presence of a serum factor (the C3 nephritic factor C3NeF) which was capable of breaking down C3 in normal serum in vitro. Metabolic studies using radioactive iodine labelled C3 showed no evidence of accelerated in vivo breakdown of parenterally administered C3 suggesting that hypocomplementaemia is either maintained by diminished C3 synthesis or that accelerated catabolism is occurring in a pool that does not freely exchange with parenterally given C3. The C3 nephritic factor has so far only been identified in patients with membranoproliferative nephritis and is therefore of major diagnostic significance in patients with glomerular disease.