Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori

Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo.     

Valproate-induced teratogenesis in Japanese rice fish (Oryzias latipes) embryogenesis

Fertilized eggs of Japanese rice fish (medaka) at three developmental stages (Iwamatsu stages 4-30) were exposed to waterborne valproic acid (VPA) (0-80 mM) in hatching solution for 48 h. The amount of valproate to cause 50% mortality (IC(50)) is found to be developmental stage-specific. The embryos were more sensitive to valproate at early stages of development (Iwamatsu stages 4-10) than in the embryos in late stages (Iwamatsu stages 17-30). Valproate exposed embryos have microcephaly and disrupted cardiovasculature with delayed vessel circulation, thrombus formation, and slow heart rate. The hatching efficiency is also reduced by valproate exposure due to developmental delay. The mRNA analysis of nine genes belong to oxidative stress (catalase, gsr, gst), neurogenesis (iro3, wnt1, shh, otx2, nlgn3b) and cell cycle regulation (ccna2) have been done. It was observed that the genes belong to oxidative stress remained unaltered after valproate exposure. However, some of the genes belong to neurogenesis (wnt1,shh, otx2 and nlgn3b) and cell cycle (ccna2) showed developmental stage-specific alteration after valproate exposure. This study indicates that valproate is able to induce some of the phenotypic features which are analogous to human fetal valproate syndrome (FVS). Modulation of genes expressed in neural tissues indicates that this fish can be used to analyze the mechanisms of many neurobehavioral disorders like Autism spectrum disorder (ASD) in human.     

Molecular analysis of the GrB mutation in Bombyx mori through the use of a chorion cDNA library

To study the molecular basis of the Gr^B mutation, which prevents the synthesis of many stage-specific chorion proteins, a cDNA library has been constructed from wild-type chorion mRNA of Bombyx mori strain 703. By differential screening of the library with +/+ and B/B mRNAs, under appropriately stringent conditions to minimize cross hybridizations of related chorion sequences, we have selected several distinct clones corresponding to RNA sequences which are affected by the mutation (that is, are represented only in +/+ mRNA) or are unaffected (that is, are represented in both +/+ and B/B mRNAs). We show by Southern analysis that, whereas unaffected gene sequences are represented in both +/+ and B/B chromosomal DNA, affected sequences have been deleted from B/B DNA. The organization and regulation of developmental stage-specific chorion genes are discussed in light of these findings and the known effects of Gr^B on stage-specific protein synthesis.     

cDNA cloning and expression of Xenopus sperm-specific basic nuclear protein 5 (SP5) gene

As part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm-specific nuclear proteins (SPs1–6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)⁺ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N-terminal regions of all the SPs(1–6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one-fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0. © 1994 Wiley-Liss, Inc.     

Gene expression in normotopic and heterotopic human bone: increased level of SP7 mRNA in pathological tissue

Head injury-induced heterotopic ossification (HO) develops at vicinity of joints and in severe cases requires surgical intervention. Our previous study demonstrated high mRNA levels of osteocalcin (OC), type 1 collagen (COL1), osteonectin and RUNX2/CBFA1 in osteocytes and lining osteoblasts from non-evolutive HO compared to equivalent healthy cells from the proximal femoral shaft of patients receiving prosthesis. This allowed a first molecular characterisation of this pathological bone. The aims of this study is to extend the analysis to 10 more genes and determine those involved in the high OC mRNA level observed in pathological bone samples. RNAs were prepared from normotopic and heterotopic human bone samples digested by collagenase. After cDNA synthesis, mRNA levels were determined by real-time PCR and normalised using beta actin and glyceraldehyde-3-phosphate dehydrogenase. OSTERIX/SP7 expression was observed for the first time in human adult bone biopsies. In HO samples higher levels of SP7 (four- to sevenfold increase) and 1alpha,25-dihydroxy vitamin D(3) receptor (VDR) (two- to threefold increase) were observed compared to control samples. Moreover, SP7 level was correlated to OC and RUNX2 levels. In control samples, OC and SP7 levels were correlated. Our study further confirms that the involvement of SP7 in bone physiology is not only limited to the developmental step. Moreover, our results support the hypothesis that in HO the high level of OC expression could be due not only to an increase in RUNX2, but also in SP7 or VDR or to an imbalance in their respective activities.     

Lunatic fringe controls T cell differentiation through modulating notch signaling

T cells differentiate from bone marrow-derived stem cells by expressing developmental stage-specific genes. We here searched arrays of genes that are highly expressed in mature CD4-CD8+ (CD8 single-positive (SP)) T cells but little in CD4+CD8+ (double-positive (DP)) cells by cDNA subtraction. Lunatic fringe (Lfng), a modulator of Notch signaling, was identified to be little expressed in DP cells and highly expressed in CD8SP T cell as well as in CD4-CD8- (double-negative (DN)) and mature CD4+CD8- (CD4SP) T cells. Thus, we examined whether such change of expression of Lfng plays a role in T cell development. We found that overexpression of Lfng in Jurkat T cells strengthened Notch signaling by reporter gene assay, indicating that Lfng is a positive regulator for Notch signaling in T cells. The enforced expression of Lfng in thymocytes enhanced the development of immature CD8SP cells but decreased mature CD4SP and CD8SP cells. In contrast, the down-regulation of Lfng in thymocytes suppressed DP cells development due to the defective transition from CD44+CD25- stage to subsequent stage in DN cells. The overexpression of Lfng in fetal liver-derived hemopoietic stem cells enhanced T cell development, whereas its down-regulation suppressed it. These results suggested that the physiological high expression of Lfng in DN cells contributes to enhance T cell differentiation through strengthening Notch signaling. Shutting down the expression of Lfng in DP cells may have a physiological role in promoting DP cells differentiation toward mature SP cells.     

Use of variability in the stage-specific transcription levels of Plasmodium falciparum in the selection of target genes

The malarial parasite Plasmodium falciparum exhibits several morphological and developmental stages. We have quantified the level of expression of a battery of genes in the ring and trophozoite stage-two of the most prominent stages in the erythrocytic development of the parasite. Using optimized RT-PCR, we observed that some of the genes show a large variation in stage-specific expression. We have also correlated the level of mRNA expression (of the target enzyme) to its metabolic requirement using specific inhibitors. This protocol gives us a handle to identify vulnerable target genes that could be used to develop antimalarials.     

Triggers and tricks of Plasmodium sexual development

Irrespective of the tremendous suffering caused by malaria, a Plasmodium infection by pathogenic blood stages is only transient and an obligate step toward the Anopheles vector where sexual reproduction and genetic recombination of the unicellular parasite takes place. Recent expression profiling studies identified the molecular make-up of female and male gametocytes. Differential promoters and translational repression through mRNA binding by a female-specific helicase help to fine-tune the expression of these sexual stage-specific genes. However, we are only just beginning to discover the triggers that initiate gametocytogenesis and the developmental programs that drive sexual development.     

Flavin-containing monooxygenase isoform 2: developmental expression in fetal and neonatal rabbit lung

Mammalian flavin-containing monooxygenase functions in the oxygenation of numerous xenobiotics containing a soft nucleophile, usually a nitrogen or sulfur. A total of five distinct flavin monooxygenase (FMO) isoforms are expressed in mammals. Individual isoforms are expressed in a sex-, age-, and tissue-specific fashion. In this study, we document the early developmental appearance of the major isoform in rabbit lung, FMO2. FMO2 catalytic activity as well as protein and mRNA are not only present in fetal and neonatal lung but, in some instances, approach levels found in the adult. The expression pattern of FMO2 is similar to that of the two major constitutive cytochromes P450 found in rabbit lung, 2B4 and 4B1. The early developmental appearance of these monooxygenases indicate an important role in the protection of the fetus and neonate against toxic insult from foreign chemicals.