Targeting of asialofetuin sugar chain-bearing liposomes to liver lysosomes

Specific direction of liposomes bearing an asialofetuin sugar chain (AFSC) to liver parenchymal cells was examined both in vivo and in vitro. The AFSC-bearing liposomes were preferentially recovered in the liver within several minutes after an intravenous injection into mice and were found to be predominantly localized in mitochondrial-lysosomal fraction. The massive distribution of the AFSC-liposomes in this fraction was also confirmed by using a lysosomal protease inhibitor, E-64-d. In isolated rat hepatocytes, the uptake of AFSC-liposomes was increased 2-3-fold as compared with the control liposomes without AFSC. Thus liposomes bearing AFSC would be useful to target enzymes to liver lysosomes.     

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.     

Proteomic studies of isoforms of the P25 component of Bombyx mori fibroin

It is recognized that P25 is one of three polypeptide components of the fibroin synthesized in the larval silk gland (SG) of silkworm, having two glycosylated isoforms. In the present study, however, eight P25 isoforms were separated by proteomics, including two-dimensional gel electrophoresis of whole SG proteins, and were identified by the peptide mass fingerprinting method. Four of the eight isoforms were identified as Bombyx mandarina P25s, although the SG of Bombyx mori has never been considered to contain the P25 from B. mandarina. It is suggested that this diversity of P25 isoforms depends on phosphorylation modification in addition to glycosylation.     

Albino (al) is a tetrahydrobiopterin (BH4)-deficient mutant of the silkworm Bombyx mori

Albino (al) is a lethal mutant of Bombyx mori that exhibits a colourless cuticle after the first ecdysis and dies without feeding on mulberry. Previous studies have indicated that sclerotisation was insufficient because of defective phenylalanine and tyrosine metabolism in albino larvae. However, the genetic mechanism underlying the albino phenotype has not been determined. Dopamine plays a central role in insect cuticle colouration and sclerotisation. The pathway for dopamine biosynthesis from phenylalanine involves phenylalanine hydroxylase (PAH; EC 1.14.16.1) and tyrosine hydroxylase (TH; EC 1.14.16.2). Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases, including PAH and TH. Thus, BH4 is indispensable for cuticle colouration and sclerotisation. Here we report on identifying mutations in the gene that encodes for the Bombyx homolog of 6-pyruvoyl-tetrahydropterin synthase (PTS) which is involved in the biosynthesis of BH4, in 2 strains with different al alleles. In strain a60 (al), a transposable element was inserted in exon 2 of BmPTS. In strain a61 (al²), an 11-bp deletion was identified in the exon 2 region of BmPTS. After oral administration of BH4 to the al² larvae, the survival rate was effectively increased and the larval integument was pigmented. These results indicated that BmPTS was responsible for the albino mutants of B. mori. We conclude that (i) a mutation in BmPTS leads to an insufficient supply of BH4 and results in defective dopamine biosynthesis and (ii) lack of dopamine results in cuticle colouration and sclerotisation failure. Lemon (lem) is a BH4-deficient mutant. It has been reported that de novo synthesis of zygotic BH4 was indispensable for viability of the embryo in eggs laid by lem (lem/lem^l) females. We found that lem/lem, al²/al² larvae produced by lem (lem/lem) females were viable during the first instar stage, suggesting that al²/al² embryo could synthesis BH4 by using maternally transmitted BmPTS.