Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.     

Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate

In cells, only properly folded procollagen trimers are secreted from the endoplasmic reticulum (ER), while improperly folded abnormal procollagens are retained within the ER. Ascorbic acid is a co-factor in procollagen hydroxylation, which in turn is required for trimer formation. We examined chaperone proteins which bound to procollagen in the absence of ascorbic acid, a model which mimics the human disease scurvy at the cellular level. We found that both prolyl 4-hydroxylase (P4-H)/protein disulfide isomerase (PDI) and HSP47 bound to procollagen in the absence of ascorbic acid. However, the binding of PDI to procollagen decreased when HSP47 was co-transfected, suggesting that HSP47 and PDI compete for binding to procollagen. These data indicate that P4-H/PDI and HSP47 have cooperative but distinct chaperone functions during procollagen biosynthesis.     

Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene

The novel mannose-binding rice lectin (MRL) purified by Sephadex G-50 or maltamyl Sepharose 4B affinity chromatography was not homogeneous, but the components were separated clearly by two dimensional polyacrylamide gel electrophoresis (1st; isoelectric focusing with Immobiline, 2nd; SDS-PAGE). The major spots were located at pI 4.85 and 4.74, and minor spots at pI 4.66, 4.56, and 4.44; all spots were distributed at about MW 45,000. Other faint spots were sometimes detected just below the major spots. In the western blot analysis, all the spots reacted with the monoclonal antibodies specific to MRL, which bound to MRL and inhibited the lectin activity to agglutinate rabbit erythrocytes. The proteins of the spots at pI 4.85, 4.77, 4.66, and 4.56 had lectin activity. The major proteins at pI 4.85 and 4.77 also had the common amino acid sequence at N-terminus, TLVKIGPWGGNGGSAQDISV, which is almost identical to salt and drought stress-inducible salT gene products in rice plants. High homology was also conserved in both the cDNA and the genomic clones encoding the MRL component at pI 4.85, which were selected with MRL-specific antibodies and an oligonucleotide designed from the partial amino acid sequence. All results suggest that MRL is composed of several isolectins, if not, related proteins having a common epitope and may belong to a family of stress-inducible proteins.     

Process of Egg Formation in the Female Body Cavity and Fertilization in Male Eggs of Phytoseiulus Persimilis (Acari: Phytoseiidae)

The process of egg formation in the body cavity of a phytoseiid mite, Phytoseiulus persimilis, was observed to examine fertilization of male eggs. After insemination, one of the ova at the periphery of the ovary began to expand, taking up yolk. Two pronuclei appeared in the expanded egg, located dorsally in the ovary, and yolk granules were formed gradually. After the egg became filled with yolk granules the two pronuclei fused. The egg moved via the narrow entrance at the ventral region into the oviduct, where the eggshell was formed. When the eggshell was complete, and while embryogenesis proceeded, the egg was deposited. In the meantime some ova began to expand sequentially and two joining pronuclei appeared in expanding eggs. The joining pronuclei in the first egg proved male diploidy. This is additional evidence of pseudo-arrhenotoky in this phytoseiid mite species, since the first eggs developed into males.     

Mass propagation of ornamental gentian in liquid medium

An efficient system for clonal mass propagation in liquid culture was established for the propagation of ornamental gentian. In a test of the requirements for three cytokinins [6-benzylaminopurine, N-(2-chloro-4-pyridyl)-N′-phenylurea and N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ)] in combination with 1-naphthaleneacetic acid (NAA), we found that effective propagation of shoots occurred with 0.01 mg l^–1 TDZ in a 300 ml conical flask that contained 100 ml of medium. The propagation of shoots was also affected by the concentrations of macronutrients (KNO₃, NH₄NO₃ and CaCl₂) and sucrose in Murashige and Skoog's (MS) medium, and it was influenced to some extent by the speed of agitation on an orbital shaker. The most efficient propagation of shoots was achieved in full-strength MS medium supplemented with 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose with agitation at 150 rpm. The propagation of shoots was maximal after 6 weeks of culture (140 shoots from five nodal segments in one flask). Large-scale propagation in a 5-l fermenter was attempted using 3 l of MS medium that contained 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose. More than 2,000 shoots were obtained in the fermenter in 5 weeks following the initial cultivation of five nodal segments for 6 weeks in one 300-ml flask. The shoots that had propagated in the fermenter were transferred directly to soil without prior rooting in vitro and were easily acclimatized within 1 month. Received: 7 October 1997 / Revision received: 16 January 1998 / Accepted: 30 January 1998     

Fluctuating Asymmetry and Male Mating Success in a Sphragis-Bearing Butterfly Luehdorfia japonica (Lepidoptera: Papilionidae)

The relationship between fluctuating asymmetry (FA) and mating success was studied within males of the sphragis-baring butterfly Luehdorfia japonica, which were collected at various periods during their mating season. FA was measured on the forewing and hindwing radius lengths of male butterflies. Mating frequency of males was estimated by assessing the degree of scale loss from their claspers. Males consume scales and use them to form sphragis on the female abdomen during copulation, sealing the ostium bursa for life. Age of males was scored as wing age 0 to 4 according to the wearing of the wing. FA was negatively correlated with mating frequency but positively correlated with wing age, and average FA decreased with mating season. As females have little chance to express mate choice, it is likely that FA is an indicator of male viability: symmetrical males live longer and/or fly more actively, resulting in a higher lifetime mating success compared to asymmetrical males.     

Rust fungi (Uredinales) found in Marshall Islands and Pohnpei

Uromyces wedeliae, Coleosporium plumeriae, andUredo guettardae are reported for the first time from Kwajalein Atoll, Republic of Marshall Islands, and Pohnpei, Federal States of Micronesia.