Pseudomosaicism in acatalasemic red cells visualized by fluorescent antibody technique

Zusammenfassung Im Blutausstrich lassen sich Erythrozyten von normalem und solche von stark vermindertem Katalasegehalt durch Verwendung von fluoreszierender Antikatalase unterscheiden. Mit dieser Methode konnte der früher erhobene Befund, wonach bei homozygoten Trägern des Enzymdefektes Akatalasie ein Pseudomosaizismus besteht, bestätigt werden. Bei der Untersuchung von Erythrozytenfraktionen verschiedener Dichte besteht eine Korrelation zwischen der Anzahl Retikulozyten und fluoreszierender Zellen. Dieser Befund passt zur Annahme, dass es sich bei der Katalaserestaktivität im Blut homozygoter Defektträger um eine instabile, jedoch antigenidentische Enzymvariante handelt.     

U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping

Most cases of Duchenne muscular dystrophy are caused by dystrophin gene mutations that disrupt the mRNA reading frame. Artificial exclusion (skipping) of a single exon would often restore the reading frame, giving rise to a shorter, but still functional dystrophin protein. Here, we analyzed the ability of antisense U7 small nuclear (sn)RNA derivatives to alter dystrophin pre-mRNA splicing. As a proof of principle, we first targeted the splice sites flanking exon 23 of dystrophin pre-mRNA in the wild-type muscle cell line C2C12 and showed precise exon 23 skipping. The same strategy was then successfully adapted to dystrophic immortalized mdx muscle cells where exon-23-skipped dystrophin mRNA rescued dystrophin protein synthesis. Moreover, we observed a stimulation of antisense U7 snRNA expression by the murine muscle creatine kinase enhancer. These results demonstrate that alteration of dystrophin pre-mRNA splicing could correct dystrophin gene mutations by expression of specific U7 snRNA constructs.     

Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice

Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein and an important constituent of the lysosomal membrane. Here we show that LAMP-2 deficiency in mice increases mortality between 20 and 40 days of age. The surviving mice are fertile and have an almost normal life span. Ultrastructurally, there is extensive accumulation of autophagic vacuoles in many tissues including liver, pancreas, spleen, kidney and skeletal and heart muscle. In hepatocytes, the autophagic degradation of long-lived proteins is severely impaired. Cardiac myocytes are ultrastructurally abnormal and heart contractility is severely reduced. These findings indicate that LAMP-2 is critical for autophagy. This theory is further substantiated by the finding that human LAMP-2 deficiency causing Danon's disease is associated with the accumulation of autophagic material in striated myocytes.     

Heterogeneity of catalase in blood of heterozygous gases of acatalasia

Zusammenfassung Mittels Chromatographie auf Calciumphosphat-DEAE-Cellulose lassen sich aus Hämolysat von Menschenerythrocyten zwei Fraktionen gereinigter Katalase darstellen. Im Gegensatz zu den Fraktionen aus normalen und homozygot akatalatischen Zellen zeigen die aus Blut heterozygoter Defektträger isolierten Katalasefraktionen ein elektrophoretisch verschiedenes Verhalten. Die Beweglichkeit der rascher wandernden Fraktion entspricht jener des normalen Enzyms, während die langsamer wandernde Fraktion sich gleich verhält wie das aus Akatalasieerythrocyten isolierte Enzym. Daraus folgt, dass zwischen Katalase in normalen und Akatalasiezellen trotz nachgewiesener Antigenidentität gewisse Strukturunterschiede zu bestehen scheinen.     

Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.     

Germline epimutation of MLH1 in individuals with multiple cancers

Epigenetic silencing can mimic genetic mutation by abolishing expression of a gene. We hypothesized that an epimutation could occur in any gene as a germline event that predisposes to disease and looked for examples in tumor suppressor genes in individuals with cancer. Here we report two individuals with soma-wide, allele-specific and mosaic hypermethylation of the DNA mismatch repair gene MLH1. Both individuals lack evidence of genetic mutation in any mismatch repair gene but have had multiple primary tumors that show mismatch repair deficiency, and both meet clinical criteria for hereditary nonpolyposis colorectal cancer. The epimutation was also present in spermatozoa of one of the individuals, indicating a germline defect and the potential for transmission to offspring. Germline epimutation provides a mechanism for phenocopying of genetic disease. The mosaicism and nonmendelian inheritance that are characteristic of epigenetic states could produce patterns of disease risk that resemble those of polygenic or complex traits.