Hiroyuki Izu

The expression profile of microRNAs in mouse embryos

MicroRNAs (miRNAs), which are non-coding RNAs 18–25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering ∼80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

Molecular Cloning, Expression, and Sequence Analysis of the Endoglycoceramidase II Gene from Rhodococcus Species Strain M-777

Endoglycoceramidase (EGCase (EC 3.2.1.123)) is a hydrolase that hydrolyzes the linkage between the oligosaccharide and ceramide of various glycosphingolipids. This paper describes the molecular cloning and expression of EGCase II, one of the isoforms of EGCases. The gene encoding EGCase II was obtained by screening of a genomic DNA library from Rhodococcus sp. strain M-777 constructed in pUC19 with oligonucleotide probes deduced from a partial amino acid sequence of the enzyme protein. RecombinantEscherichia coli cells in which the EGCase II gene was expressed produced 14 units of the enzyme per liter of culture medium but did not produce sphingomyelinase. Recombinant EGCase II was a functioning enzyme with substrate specificity identical to that of the wild-type enzyme. Sequence analysis showed the presence of an open reading frame of 1470 base pairs encoding 490 amino acids. The N-terminal region of the deduced amino acid sequence had the general pattern of signal peptides of secreted prokaryotic proteins. Interestingly, the consensus sequence in the active site region of the endo-1,4-β-glucanase family A was found in the amino acid sequence of EGCase II.

Enzymatic N-deacylation of sphingolipids

Publisher Summary Lysosphingolipids, sphingolipids with an N-deacylated ceramide moiety, are present at low levels in normal tissues, but accumulate abnormally in cells in various lysosomal storage diseases. For example, in Krabbes disease, caused by a deficiency of β -galactosylceramidase, abnormal accumulation of galactosylceramide as well as its lyso form is observed. Lyso-GM2 and lysosphingomyelin have been detected in the brain of patients with Tay-Sachs and Niemann-Pick type-A disease, respectively, whereas they are barely detectable in the normal brain. Lysosphingolipids inhibit protein kinase C, which could be responsible for the pathogenesis of sphingolipidoses. Several lines of evidence have suggested the biological significance of lysosphingolipids in various cell activities. Lysosphingolipids are useful for preparing sphingolipid derivatives containing appropriately labeled fatty acids and can be coupled with either appropriate proteins or gel matrix for affinity columns utilizing the amino groups newly generated in lysosphingolipids. This chapter describes a novel enzyme, tentatively designated sphingolipid ceramide N-deacylase (SCDase), which is capable of cleaving the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms. To date, the preparation of lysosphingolipids has been performed using purely chemical procedures, which are somewhat troublesome, time-consuming, and give a low yield. Using the SCDase we were able to obtain easily the lyso forms of all species of glycosphingolipids and sphingomyelin without any alternation of their polar portions and sphingoid moieties.