Yunis-Varon syndrome: evidence for a lysosomal storage disease

We present clinical and neuropathological findings in a female infant with Yunis-Varon syndrome (YVS) comprising absence of thumbs and halluces, aphalangia of fingers and toes, hypoplasia of clavicles, severely undermineralized skeleton (especially skull), microcephaly, and multiple nonskeletal anomalies. The patient also had a Dandy-Walker malformation, hydrocephalus, and hypertension, which were not reported previously in YVS. The infant excreted an abnormal unidentified oligosaccharide. The patient died at day 108 with severe neurological impairment. Autopsy showed prominent intraneuronal inclusions with vacuolar degeneration, mainly in the thalamic, dentate nuclei, cerebellar cortex, and inferior olivary nuclei. No storage phenomena were observed in other tissues. These findings strongly suggest that a lysosomal storage disorder is involved in the pathogenesis of Yunis-Varon syndrome.     

Mutations causing aspartylglucosaminuria (AGU): a lysosomal accumulation disease.

This article provides a review of the mutations reported so far in the lysosomal storage disease aspartylglucosaminuria (AGU). The clinical symptoms, biochemical findings, and diagnostic possibilities of the disease are introduced. The prevalence and biological consequences of the found mutations are then described, as well as the availability of a new rapid DNA test suitable for carrier screening. This test will be especially applicable in the genetically isolated Finnish population, where the carrier frequency of AGU was found to be as high as 1:36. Finally, future prospects dealing with the foreseeable therapeutic interventions of the disease are discussed.     

Beta-mannosidosis mice: a model for the human lysosomal storage disease

Beta-mannosidase, a lysosomal enzyme which acts exclusively at the last step of oligosaccharide catabolism in glycoprotein degradation, functions to cleave the unique beta-linked mannose sugar found in all N-linked oligosaccharides of glycoproteins. Deficiency of this enzyme results in beta-mannosidosis, a lysosomal storage disease characterized by the cellular accumulation of small oligosaccharides. In human beta-mannosidosis, the clinical presentation is variable and can be mild, even when caused by functionally null mutations. In contrast, two existing ruminant animal models have disease that is consistent and severe. To further explore the molecular pathology of this disease and to investigate potential treatment strategies, we produced a beta-mannosidase knockout mouse. Homozygous mutant mice have undetectable beta-mannosidase activity. General appearance and growth of the knockout mice are similar to the wild-type littermates. At >1 year of age, these mice exhibit no dysmorphology or overt neurological problems. The mutant animals have consistent cytoplasmic vacuolation in the central nervous system and minimal vacuolation in most visceral organs. Thin-layer chromatography demonstrated an accumulation of disaccharide in epididymis and brain. This mouse model closely resembles human beta-mannosidosis and provides a useful tool for studying the phenotypic variation in different species and will facilitate the study of potential therapies for lysosomal storage diseases.     

Angiogenesis and pulmonary hypertension: a unique process in a unique disease

Severe pulmonary hypertension is a fatal condition associated with marked alterations of the cellular components of pulmonary arteries. In this review, we discuss the component of endothelial cell proliferation present in pulmonary arteries in patients with severe pulmonary hypertension. Because these proliferated endothelial cells exhibit markers of angiogenesis, we have named this process as "disordered or misguided angiogenesis." We also discuss the recent evidence that germline or somatic inactivating mutations in tumor suppressor or proapoptotic genes may play a significant role in the abnormal proliferation of pulmonary endothelial cells. The unraveling of the pathobiology of severe pulmonary hypertension may lead us to novel therapies and approaches to better diagnose the disease.     

Yunis‐Varon syndrome: Evidence for a lysosomal storage disease

We present clinical and neuropathological findings in a female infant with Yunis‐Varon syndrome (YVS) comprising absence of thumbs and halluces, aphalangia of fingers and toes, hypoplasia of clavicles, severely undermineralized skeleton (especially skull), microcephaly, and multiple nonskeletal anomalies. The patient also had a Dandy‐Walker malformation, hydrocephalus, and hypertension, which were not reported previously in YVS. The infant excreted an abnormal unidentified oligosaccharide. The patient died at day 108 with severe neurological impairment. Autopsy showed prominent intraneuronal inclusions with vacuolar degeneration, mainly in the thalamic, dentate nuclei, cerebellar cortex, and inferior olivary nuclei. No storage phenomena were observed in other tissues. These findings strongly suggest that a lysosomal storage disorder is involved in the pathogenesis of Yunis‐Varon syndrome. Am. J. Med. Genet. 95:157–160, 2000. © 2000 Wiley‐Liss, Inc.     

Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus

The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.     

Ser72Pro active-site disease mutation in human lysosomal aspartylglucosaminidase: abnormal intracellular processing and evidence for extracellular activation

Aspartylglucosaminuria (AGU) is a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (AGA). We report here a T214C mutation leading to a Ser72Pro substitution in four Arab families. This is the first naturally occurring AGU mutation involving an active-site amino acid of this recently crystallized hydrolase and it seems to represent the second most common AGU mutation worldwide. The intracellular consequences of the Ser72Pro mutation were analyzed by transient expression in COS-1 cells and we were able to demonstrate that this active-site mutation most probably does not destroy the enzyme activity per se, but specifically prevents the proteolytic activation cleavage of AGA in the endoplasmic reticulum (ER). The mutant enzyme is, however, folded correctly enough to allow mannose-6- phosphorylation and targeting to lysosomes. The overexpressed mutant enzyme remained inactive intracellularly, but the secreted mutant precursor was proteolytically activated extracellularly, resulting in a similar subunit composition to that in the wild-type AGA in the ER. The partially activated mutant enzyme was endocytosed further by the recipient cells. These data demonstrate that the proteolytic activation of AGA can also occur extracellularly and suggest that the driving mechanism of AGA precursor cleavage is autocatalytic.      

The cell cycle in Alzheimer disease: a unique target for neuropharmacology

Several hypotheses have been proposed attempting to explain the pathogenesis of Alzheimer disease including, among others, theories involving amyloid deposition, tau phosphorylation, oxidative stress, metal ion dysregulation and inflammation. While there is strong evidence suggesting that each one of these proposed mechanisms contributes to disease pathogenesis, none of these mechanisms are able to account for all the physiological changes that occur during the course of the disease. For this reason, we and others have begun the search for a causative factor that predates known features found in Alzheimer disease, and that might therefore be a fundamental initiator of the pathophysiological cascade. We propose that the dysregulation of the cell cycle that occurs in neurons susceptible to degeneration in the hippocampus during Alzheimer disease is a potential causative factor that, together with oxidative stress, would initiate all known pathological events. Neuronal changes supporting alterations in cell cycle control in the etiology of Alzheimer disease include the ectopic expression of markers of the cell cycle, organelle kinesis and cytoskeletal alterations including tau phosphorylation. Such mitotic alterations are not only one of the earliest neuronal abnormalities in the disease, but as discussed herein, are also intimately linked to all of the other pathological hallmarks of Alzheimer disease including tau protein, amyloid beta protein precursor and oxidative stress, and even risk factors such as mutations in the presenilin genes. Therefore, therapeutic interventions targeted toward ameliorating mitotic changes would be predicted to have a profound and positive impact on Alzheimer disease progression.     

Parallel regulation of renin and lysosomal integral membrane protein 2 in renin-producing cells: further evidence for a lysosomal nature of renin secretory vesicles

The protease renin is the key enzyme in the renin–angiotensin system (RAS) that regulates extracellular volume and blood pressure. Renin is synthesized in renal juxtaglomerular cells (JG cells) as the inactive precursor prorenin. Activation of prorenin by cleavage of the prosegment occurs in renin storage vesicles that have lysosomal properties. To characterize the renin storage vesicles more precisely, the expression and functional relevance of the major lysosomal membrane proteins lysosomal-associated membrane protein 1 (LAMP-1), LAMP-2, and lysosomal integral membrane protein 2 (LIMP-2) were determined in JG cells. Immunostaining experiments revealed strong coexpression of renin with the LIMP-2 (SCARB2), while faint staining of LAMP-1 and LAMP-2 was detected in some JG cells only. Stimulation of the renin system (ACE inhibitor, renal hypoperfusion) resulted in the recruitment of renin-producing cells in the afferent arterioles and parallel upregulation of LIMP-2, but not LAMP-1 or LAMP-2. Despite the coregulation of renin and LIMP-2, LIMP-2-deficient mice had normal renal renin mRNA levels, renal renin and prorenin contents, and plasma renin and prorenin concentrations under control conditions and in response to stimulation with a low salt diet (with or without angiotensin-converting enzyme (ACE) inhibition). No differences in the size or number of renin vesicles were detected using electron microscopy. Acute stimulation of renin release by isoproterenol exerted similar responses in both genotypes in vivo and in isolated perfused kidneys. Renin and the major lysosomal protein LIMP-2 are colocalized and coregulated in renal JG cells, further corroborating the lysosomal nature of renin storage vesicles. LIMP-2 does not appear to play an obvious role in the regulation of renin synthesis or release.     

A genomic rearrangement resulting in a tandem duplication is associated with split hand-split foot malformation 3 (SHFM3) at 10q24

Split hand-split foot malformation (SHFM) is characterized by hypoplasia/aplasia of the central digits with fusion of the remaining digits. SHFM is usually an autosomal dominant condition and at least five loci have been identified in humans. Mutation analysis of the DACTYLIN gene, suspected to be responsible for SHFM3 in chromosome 10q24, was conducted in seven SHFM patients. We screened the coding region of DACTYLIN by single-strand conformation polymorphism and sequencing, and found no point mutations. However, Southern, pulsed field gel electrophoresis and dosage analyses demonstrated a complex rearrangement associated with a approximately 0.5 Mb tandem duplication in all the patients. The distal and proximal breakpoints were within an 80 and 130 kb region, respectively. This duplicated region contained a disrupted extra copy of the DACTYLIN gene and the entire LBX1 and beta-TRCP genes, known to be involved in limb development. The possible role of these genes in the SHFM3 phenotype is discussed.     

Target structure for natural killer cells: evidence against a unique role for transferrin receptor

The transferrin receptor (TfR) was recently proposed as putative natural killer (NK) cell target structure. Here data are presented against this hypothesis and it is shown that low TfR expression and high NK sensitivity can occur concommitantly . K562 cells were studied at various stages of cell proliferation. No change in NK sensitivity could be observed between exponential growth and the plateau phase, whereas TfR expression completely disappeared during the latter. Protein synthesis inhibitors such as cycloheximide (1 microgram/ml, 48 h) and actinomycin D (50 micrograms/ml, 48 h), that abolished the TfR expression at the K562 cell surface, had no effect on NK sensitivity. Similarly, hemin induction (0.1 mM, 5 days) did not change NK susceptibility of K562 cells but considerably diminished TfR expression. Moreover, attempts to block NK sensitivity with anti-TfR monoclonal antibodies were unsuccessful, even when the 42.6 antibody, which is known to bind to the active site of TfR, was used. Finally, no blocking of NK sensitivity could be achieved when K562 cells were preincubated with saturating concentrations of transferrin or when transferrin was added during the NK assay. It therefore seems doubtful that TfR is the unique target structure for NK cells. It remains possible that TfR and NK target structures are often coexpressed on actively dividing cells.     

LPS-induced 111In-eosinophil accumulation in guinea-pig skin: evidence for a role for TNF-alpha.

Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria with powerful pro-inflammatory activities. Although the mechanisms involved in LPS-induced neutrophil accumulation have been studied extensively, few reports have focused on the effects of LPS on eosinophil infiltration. In this study we have used an in vivo model of local 111In-eosinophil accumulation in the guinea-pig to investigate the mechanisms of LPS-induced eosinophilia. Using a 4-hr in vivo test period, the intradermal injection of LPS (50-1000 ng/site) led to a marked and dose-dependent accumulation of 111In-eosinophils into guinea-pig skin sites. Time-course experiments revealed that this cell infiltration was delayed in onset, becoming significant 1 hr after the intradermal administration of LPS. The slow development of the response and its sensitivity to the locally administered protein synthesis inhibitor, actinomycin D, suggested that the LPS-induced 111In-eosinophil accumulation in vivo is mediated by the generation of de novo proteins. The intravenous pretreatment of guinea-pigs with a soluble tumour necrosis factor-alpha (TNF-alpha) receptor fusion protein (TNFR-IgG, 1 mg/kg), potently inhibited the 111In-eosinophil accumulation induced by LPS. Our results demonstrate that LPS can induce 111In-eosinophil accumulation in vivo in guinea-pig skin, and that this process is mediated by TNF-alpha.     

Rapid pathotyping of Newcastle disease virus using a single-chain Fv displayed on phage against the C-terminal end of the F2 polypeptide

Filamentous bacteriophage display technology has been used to generate specific antibody fragments for differentiating virulent and avirulent Newcastle disease virus. A single-chain Fv fragment to the motif (112)RRQ(114), present at the F2 C-terminal end of many virulent Newcastle disease virus isolates, was isolated from a phage display library derived from a rabbit immunized with a peptide conjugate. An ELISA evaluation was carried out to test its ability to differentiate between 11 avirulent and 34 virulent NDV isolates. The antibody fragment reacted with 25/28 virulent viruses with the putative motif (112)RRQ(114). The three exceptions were viruses with an arginine instead of glycine, at position 110 of the fusion protein, just preceding the cleavage site. Five of six virulent isolates, whose predicted motif was different from that usually found in virulent strains, also tested negative. However, the antibody did react with one isolate with the motif (112)KRQ(114). There was no apparent reactivity with any of the avirulent isolates tested. We conclude that this antibody may, in the future, be a useful aid for the pathotyping of NDV isolates.     

Deletion in the C-terminal domain of ClpX delayed entry of Salmonella enterica into a viable but non-culturable state

Under stressful conditions, bacteria enter a viable but non-culturable (VBNC) state in which they are alive but fail to grow on conventional media. The molecular basis underlying this state is unknown. To identify the key gene responsible for the VBNC state in Salmonella spp., we examined a S. Typhimurium LT2 VBNC mutant, which shows a characteristic delay in entering the VBNC state. The mutant showed a higher level of expression of general stress sigma factor RpoS than wild-type LT2. The mutant carried a 99-bp in-frame deletion in the clpX gene (clpX_Δ323–355). ClpX is known to form a ClpXP protease complex with ClpP, which plays a role in the degradation of RpoS. To investigate the effect of clpX_Δ323–355 on VBNC induction, ΔclpX and clpX_Δ323–355 strains were generated from LT2 cells. Compared to LT2, the ΔclpX and clpX_Δ323–355 strains showed greater amounts of RpoS and required a longer incubation time for induction into the VBNC state. These results suggest that residues 323–355 of ClpX play a major role in the hexameric formation or function of ClpX and in the rate of induction of the VBNC state.     

Mindfulness meditation-related pain relief: evidence for unique brain mechanisms in the regulation of pain

The cognitive modulation of pain is influenced by a number of factors ranging from attention, beliefs, conditioning, expectations, mood, and the regulation of emotional responses to noxious sensory events. Recently, mindfulness meditation has been found attenuate pain through some of these mechanisms including enhanced cognitive and emotional control, as well as altering the contextual evaluation of sensory events. This review discusses the brain mechanisms involved in mindfulness meditation-related pain relief across different meditative techniques, expertise and training levels, experimental procedures, and neuroimaging methodologies. Converging lines of neuroimaging evidence reveal that mindfulness meditation-related pain relief is associated with unique appraisal cognitive processes depending on expertise level and meditation tradition. Moreover, it is postulated that mindfulness meditation-related pain relief may share a common final pathway with other cognitive techniques in the modulation of pain.     

Electron microscopy in end stage renal disease: a case of Fabry's disease

Fabry's disease is an X-linked error of metabolism with deficiency of the enzyme α-glycosidase A, and glycosphingolipid accumulation in multiple tissues. Patients may be asymptomatic and present with advanced disease. We report a case of a 38 year old white male who presented with end stage renal disease of unknown etiology. He received a living-related donor kidney transplant (mother), but lost the graft after 10 years to multiple episodes of rejection. Review of the native renal biopsy with added ultrastructural studies established the diagnosis of Fabry's disease. Evaluation of renal biopsies showing advanced chronic injury should include electron microscopic studies, which may reveal characteristic diagnostic features, as seen in this case of Fabry's disease. Identification of hereditary disorders involving the kidney is important for appropriate treatment and prevention of disease recurrence. Potential living related donors should be screened for genetic involvement.     

Ku acts in a unique way at the mammalian telomere to prevent end joining

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.