Morphological and histological examination of polyphenic wing formation in the pea aphid <Emphasis Type="Italic">Acyrthosiphon pisum</Emphasis> (Hemiptera,Hexapoda)

Aphids display divergent adult phenotypes, depending on environmental conditions experienced during their embyonic and nymphal stages in their complex life cycles. The plastic developmental mode is an extreme case of phenotypic plasticity, so-called “polyphenism”, in which discrete multiple phenotypes are produced based on a single genome. For example, winged and wingless adult females are derived from a single genotype. However, the developmental mechanisms producing these polyphenic traits according to the extrinsic stimuli, such as density conditions, still remain unknown. In this study, to analyze the developmental processes underlying the wing polyphenism, we extensively observed and compared wing development in the winged and wingless individuals in parthenogenetic generations of the aphid Acyrthosiphon pisum (Harris), using scanning electron microscopy and histological sectioning. At the first-instar stage, the wing primordia were observed both in the future winged (W) and wingless (WL) nymphs. Developmental differences can be seen from the second-instar stage, when wing primordia degenerate in the WL nymphs, while they develop and become more thickened in the W nymphs, suggesting that the developmental programs should be launched prior to this stage. Furthermore, during the third- to fifth-instar stages, wing buds and flight muscles were well developed in the W nymphs, while wing primordia completely disappeared in the WL ones. In addition, the observation on the detailed developmental process of wing primordia during the third-instar W nymphs showed that the wing buds become swollen especially at the basal part, even during the intermolt period. This was caused by the development of wing epithelia under the cuticle of this instar nymph. Actually on the surface of the cuticle of wing-bud bases, there were numerous furrows, which gradually expand during the intermolt period. The similar situation was also observed at the forth-instar nymphs, in which the wings are formed in the complicated manner inside the wing pads. Furthermore, the developmental process of flight muscles was also described in detail. These dynamic developmental differences between the wing morphs should be regulated under the gene expression cascades that switch according to environmental stimuli.     

The chromosomal normality of in vitro-fertilized rabbit oocytes

The chromosomal normality of rabbit oocytes fertilized in vitro was examined. Ovum donors were superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Follicular oocytes were collected laparoscopically 9-10 h after hCG treatment and incubated in vitro with spermatozoa capacitated in vivo. Of 267 aspirated oocytes, 191 (71.4%) were fertilized in vitro and developed to the 2- to 8-cell stage 24-48 h after insemination. In the chromosomal studies, 121 (63.4%) were examined. Of these, 94 (77.7%) had a normal diploid complement of chromosomes (2n = 44) and 6 (5%) showed aneuploidy. Of the remaining 21, 20 were triploid and one was tetraploid. The incidence of triploid oocytes after in vitro fertilization was higher than the rate in vivo (16.5% vs. 9.0%, respectively). These triploid oocytes were suspected to be the result of polyspermic fertilization in vitro. In addition, at the Metaphase II stage, 62 (89.9%) of 69 induced, preovulatory oocytes had a normal number of chromosomes.     

A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis

The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.     

Isolation and Characterization of Temperature-Sensitive Mutations in the Ras2 and Cyr1 Genes of Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.     

E-Cell 2: multi-platform E-Cell simulation system

A new version of the E-Cell simulation system,which runs on Windows as well as Linux, has been released as free software under the terms of the GNU General Public License.     

Purification of fetal mouse hepatoblasts by magnetic beads coated with monoclonal anti-e-cadherin antibodies and their in vitro culture

A simple, rapid, and reproducible method of fetal hepatoblast purification was established to investigate mechanisms controlling interactions between hepatoblasts and nonparenchymal cells during liver development. Because E-cadherin is exclusively expressed on the cell membrane of hepatoblasts, magnetic beads coated with monoclonal antibodies to an extracellular epitope of its molecule were used to purify hepatoblasts from a cell suspension prepared from 12.5-day fetal mouse livers. The purity and yield in the hepatoblast fraction prepared in our protocol were more than 90% and approximately 30%, respectively. The nonparenchymal fraction rarely contained hepatoblasts; the rate of hepatoblast contamination in this fraction was less than 1%. Separate cultures of these two fractions were compared with cocultures of both fractions. In culture of the hepatoblast fraction, hepatoblasts formed aggregates similar to a bunch of grapes via their loose adhesion, floating in the medium after 24 h, and dissociated into single cells from the aggregates after 120 h of culture. By contrast, in the mixed culture, the majority of hepatoblasts formed multicellular spheroids after 24 h, and these spheroids changed into monolayer cell sheets after 120 h of culture. The cells comprising these monolayer sheets abundantly expressed albumin and carbamoylphosphate synthase I. In the mixed culture, fibroblastic cells also proliferated extensively with spreading on glass slides and surrounded the hepatoblast or hepatocyte colonies. On the other hand, fibroblastic cells spreading on glass slides decreased gradually in cultures of the nonparenchymal cell fraction alone. These findings indicated that the coexistence of hepatoblasts and nonparenchymal cells may be essential for their mutual survival, proliferation, differentiation, and morphogenesis. The conditioned medium of fetal liver cell cultures could partially replace the effects of the nonparenchymal cells on hepatoblasts in vitro. Our isolation protocol for fetal mouse hepatoblasts using immunobeads can greatly facilitate studies on mechanisms of cell-cell interactions during liver development.     

Deletion of a chaperonin 60 beta gene leads to cell death in the Arabidopsis lesion initiation 1 mutant

Lesion mimic mutants develop spontaneous cell death without pathogen attack. Some of the genes defined by these mutations may function as regulators of cell death, whereas others may perturb cellular metabolism in a way that leads to cell death. To understand the molecular mechanism of cell death in lesion mimic mutants, we isolated a lesion initiation 1 (len1) mutant by a T-DNA tagging method. The len1 mutant develops lesions on its leaves and expresses systemic acquired resistance (SAR). LEN1 was identified to encode a chloroplast chaperonin 60 beta (Cpn60 beta), a homologue of bacterial GroEL. The recombinant LEN1 had molecular chaperone activity for suppressing protein aggregation in vitro. Moreover, len1 plants develop accelerated cell death to heat shock stress in comparison with wild-type plants. The chlorophyll a/b binding protein (CAB) was present in len1 plants at a lower level than in the wild-type plants. These results indicate that LEN1 functions as a molecular chaperone in chloroplasts and its deletion leads to cell death in Arabidopsis.     

Immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes

By the use of electron immunoperoxidase cytochemistry at the ultrastructural level, the relationship of the surrounding sac of the autophagic vacuoles to the different cytomembranes was studied. When the endoplasmic reticulum was completely stained for microsomal carboxyesterase E1, the enzyme was not found to be labeled in the developed envelopes forming autophagic vacuoles. The autophagic envelope at the formative stages was also devoid of albumin which intensely stained Golgi cisternae. However, although it was rare, the endoplasmic reticulum showed an electron-lucent region like an early autophagic envelope in its cisternae which was lacking in carboxyesterase E1. In addition, deeply curving swelled cisternae where carboxyesterase E1 was found at the edges were occasionally encountered. These observations suggest that the segregating membranes arise from an endoplasmic reticulum and the structural characteristics of the endoplasmic membranes change at very early stages of formation of autophagic vacuoles. Acid phosphatase, a lysosomal marker enzyme, began to be localized on sections of the double membranes of newly created autophagic vacuoles. The enzyme spread all along the limiting membranes of the autophagic vacuoles, while, at the same time, the double membranes were converted into a single membrane. A lysosomal membrane glycoprotein (LGP107) was also localized on the surrounding envelope of autophagic vacuoles in a fashion similar to that of acid phosphatase. Lysosomal hydrolases seem to play some role in the conversion of double limiting membranes into a single limiting membrane.     

Structure of human peptidyl‐prolyl cis–trans isomerase FKBP22 containing two EF‐hand motifs

The FK506‐binding protein (FKBP) family consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. It is assumed that all members are peptidyl‐prolyl cis–trans isomerases with the enzymatic function attributed to the FKBP domain. Six members of this family localize to the mammalian endoplasmic reticulum (ER). Four of them, FKBP22 (encoded by the FKBP14 gene), FKBP23 (FKBP7), FKBP60 (FKBP9), and FKBP65 (FKBP10), are unique among all FKBPs as they contain the EF‐hand motifs. Little is known about the biological roles of these proteins, but emerging genetics studies are attracting great interest to the ER resident FKBPs, as mutations in genes encoding FKBP10 and FKBP14 were shown to cause a variety of matrix disorders. Although the structural organization of the FKBP‐type domain as well as of the EF‐hand motif has been known for a while, it is difficult to conclude how these structures are combined and how it affects the protein functionality. We have determined a unique 1.9 Å resolution crystal structure for human FKBP22, which can serve as a prototype for other EF hand‐containing FKBPs. The EF‐hand motifs of two FKBP22 molecules form a dimeric complex with an elongated and predominantly hydrophobic cavity that can potentially be occupied by an aliphatic ligand. The FKBP‐type domains are separated by a cleft and their putative active sites can catalyze isomerazation of two bonds within a polypeptide chain in extended conformation. These structural results are of prime interest for understanding biological functions of ER resident FKBPs containing EF‐hand motifs.