Functions of Heparan Sulfate Proteoglycans in Development: Insights From Drosophila Models.

Heparan sulfate proteoglycans (HSPGs) are a class of carbohydrate-modified proteins involved in key biological processes, including growth factor signaling, cell adhesion, and enzymatic catalysis. HSPGs serve as coreceptors for a number of ligand molecules to regulate their signaling and distribution. These HS-dependent factors include fibroblast growth factors, bone morphogenetic proteins, Wnt-related factors, hedgehog, and cytokines. Several classes of HSPGs are evolutionarily conserved from humans to the genetically tractable model organism Drosophila. Sophisticated molecular genetic tools available in Drosophila provide for a powerful system to address unanswered questions regarding in vivo functions of HSPGs. These studies have highlighted the functions of HSPGs in the regulation of significant developmental events, such as morphogen gradient formation, nervous system formation, and the stem cell niche. Drosophila genetics has also established HSPGs as key factors in feedback controls that ensure robustness in developmental systems.

Regulation of dally, an integral membrane proteoglycan, and its function during adult sensory organ formation of Drosophila.

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. Here, we show that dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. We have also determined that, for pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters.

Transcriptional and posttranscriptional regulation of the gene for Dally, a Drosophila integral membrane proteoglycan.

"division abnormally delayed"(dally), a Drosophila member of the glypican family, has been implicated in Dpp and Wg signaling. Here, we report the genomic structure and regulation of the dally gene. The dally gene is composed of nine exons, and its expression is controlled by a TATA-less promoter. Analysis of transgenic flies bearing the dally promoter fused to the lacZ reporter gene showed that a 371 bp sequence of the dally 5' flanking region was capable of mimicking the patterns of dally enhancer trap expression in developing tissues, including embryonic epidermis and imaginal discs. The tissue-specific enhancers that drive marker gene expression in embryo and the wing disc are mapped in the 5' upstream region of dally gene. We propose that dally gene expression is also regulated posttranscriptionally by controlling the translation efficiency and stability of its mRNA.

Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST) gene. Structure, expression, and function in the formation of the tracheal system.

Heparan sulfate, one of the most abundant components of the cell surface and the extracellular matrix, is involved in a variety of biological processes such as growth factor signaling, cell adhesion, and enzymatic catalysis. The heparan sulfate chains have markedly heterogeneous structures in which distinct sequences of sulfate groups determine specific binding properties. Sulfation at each different position of heparan sulfate is catalyzed by distinct enzymes, sulfotransferases. In this study, we identified and characterized Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST). The deduced primary structure of dHS6ST exhibited several common features found in those of mammalian HS6STs. We confirmed that, when the protein encoded by the cDNA was expressed in COS-7 cells, it showed HS6ST activity. Whole mount in situ hybridization revealed highly specific expression of dHS6ST mRNA in embryonic tracheal cells. The spatial and temporal pattern of dHS6ST expression in these cells clearly resembles that of the Drosophila fibroblast growth factor (FGF) receptor, breathless (btl). RNA interference experiments demonstrated that reduced dHS6ST activity caused embryonic lethality and disruption of the primary branching of the tracheal system. These phenotypes were reminiscent of the defects observed in mutants of FGF signaling components. We also show that FGF-dependent mitogen-activated protein kinase activation is significantly reduced in dHS6ST double-stranded RNA-injected embryos. These findings indicate that dHS6ST is required for tracheal development in Drosophila and suggest the evolutionally conserved roles of 6-O-sulfated heparan sulfate in FGF signaling.

Structural analysis of gene encoding cuticle protein BMCP18, and characterization of its putative transcription factor in the silkworm, Bombyx mori.

BMCP18(2) is one of the major cuticle proteins identified in the larval cuticle of the silkworm, Bombyx mori. A genomic clone coding for BMCP18 was isolated from a B. mori genomic library, and its structure was analyzed. The BMCP18 gene consists of three exons interspersed by two introns. Bm1 element-like sequences were identified around this gene, suggesting possible involvement of this retroposon in the duplication of B. mori cuticle protein genes during evolution. A structural comparison of the BMCP18 gene and related cuticle protein genes of other lepidopteran species (MSCP14.6 and HCCP12) showed that the 5' upstream region of the BMCP18, MSCP14.6, and HCCP12 genes has a 12-bp identical sequence matching the recognition sequence for transcription factors COUP-TF and HNF-4. This implies that molecular mechanisms regulating expression of these cuticle protein genes are also conserved. mRNAs coding for Bmsvp, the B. mori homolog of Drosophila Seven-up, which is known as a homolog of vertebrate COUP-TF, and BmHNF-4, a homolog of vertebrate HNF-4, were detected in the larval epidermis. Bmsvp bound to the 12-bp sequence in vitro, suggesting that Bmsvp regulates the BMCP18 gene expression.

Definition of crucial structural factors of acetogenins, potent inhibitors of mitochondrial complex I.

Some natural acetogenins are the most potent inhibitors of bovine heart mitochondrial complex I. These compounds are characterized by two functional units (i.e. hydroxylated tetrahydrofuran (THF) and alpha,beta-unsaturated gamma-lactone ring moieties) separated by a long alkyl spacer. To elucidate which structural factors of acetogenins including their active conformation are crucial for the potent inhibitory effect, we synthesized a series of novel acetogenin analogues possessing bis-THF rings. The present study clearly demonstrated that the natural gamma-lactone ring is not crucial for the potent inhibition, although this moiety is the most common structural unit among a large number of natural acetogenins and has been suggested to be the only reactive species that directly interacts with the enzyme (Shimada et al., Biochemistry 37 (1998) 854-866). The presence of free hydroxy group(s) in the adjacent bis-THF rings was favorable, but not essential, for the potent activity. This was probably because high polarity (or hydrophilicity), rather than hydrogen bond-donating ability, around the bis-THF rings is required to retain the inhibitor in the active conformation. Interestingly, length of the alkyl spacer proved to be a very important structural factor for the potent activity, the optimal length being approximately 13 carbon atoms. The present study provided further strong evidence for the previous proposal (Kuwabara et al., Eur. J. Biochem. 267 (2000) 2538-2546) that the gamma-lactone and THF ring moieties act in a cooperative manner on complex I with the support of some specific conformation of the spacer.

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.

The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila.

Wingless (Wg) is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. We demonstrate here that the protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled (fz)/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. We also show that dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes.

Molecular cloning and characterization of a cDNA encoding a larval cuticle protein of Bombyx mori.

Cuticle proteins termed LCPs are the major protein components of the larval integument of the silkworm, Bombyx mori. We purified an 18 kDa LCP (LCP18) from the guanidine hydrochloride extract of the larval cuticle and identified an LCP18 cDNA clone. The deduced primary structure and mRNA expression pattern of LCP18 are similar to those of other Bombyx LCPs and to several cuticle proteins identified in other insect species. RNA blot analysis demonstrated that the biosynthesis of LCP18 is regulated in a stage-dependent manner at the level of mRNA in epidermal cells. An in vivo study using a juvenile hormone analogue suggested that juvenile hormone positively regulates expression of LCP18 mRNA during larval intermolt stages.

Structure and expression of a cyst specific protein of Acanthamoeba castellanii.

The life cycle of Acanthamoeba is divided into a growth-division phase and two distinctive processes of cellular differentiation, termed encystment and excystment. Polyacrylamide gel electrophoresis revealed that a specific protein of 21 kDa in molecular weight occurs in the cyst, but not in the trophozoite stages of A. castellanii Neff strain. This cyst-specific protein, designated as CSP21, was purified from guanidine-HCl extract of cyst wall and anti-CSP21 antibody was produced. Immunoblotting of proteins extracted from a variety of species of Acanthamoeba genus suggested that the antibody is specific for group II amoebae, therefore, providing a useful tool for Acanthamoebae taxonomy. A cDNA clone for A. castellanii CSP21 was isolated by immunoscreening of a cDNA expression library constructed from mRNA of amoebae at encysting stage. The deduced primary structure indicated that CSP21 is a hydrophilic protein showing no significant homology with peptides thus far published. RNA blot analysis showed that the expression of CSP21 mRNA was restricted within early stages of encystment, suggesting that the biosynthesis of CSP21 is regulated at mRNA level.

dally, a Drosophila glypican, controls cellular responses to the TGF-beta-related morphogen, Dpp.

Decapentaplegic (Dpp) is a Drosophila member of the Transforming Growth Factor-beta (TGF-beta)/Bone Morphogenetic Protein (BMP) superfamily of growth factors. Dpp serves as a classical morphogen, where concentration gradients of this secreted factor control patterning over many cell dimensions. Regulating the level of Dpp signaling is therefore critical to its function during development. One type of molecule proposed to modulate growth factor signaling at the cell surface are integral membrane proteoglycans. We show here that division abnormally delayed (dally), a Drosophila member of the glypican family of integral membrane proteoglycans is required for normal Dpp signaling during development, affecting cellular responses to this morphogen. Ectopic expression of dally+ can alter the patterning activity of Dpp, suggesting a role for dally+ in modulating Dpp signaling strength. These findings support a role for members of the glypican family in controlling TGF-beta/BMP activity in vivo by affecting signaling at the cell surface.

Purification and cDNA cloning of evolutionally conserved larval cuticle proteins of the silkworm, Bombyx mori.

A specific set of structural proteins termed larval cuticle proteins (LCPs) accumulates in integuments during larval development of the silkworm, Bombyx mori. Two major larval cuticle proteins, LCP17 and LCP22, were purified from the guanidine hydrochloride extract of the larval cuticle, and specific antibodies were raised against these proteins. Immunoblot analysis revealed that both LCPs are actively synthesized during larval intermolt stages, whereas the LCP17 epitope is also slightly but significantly detectable in pupal integuments. cDNA clones for LCPs were isolated by immunoscreening of the cDNA expression library constructed from larval epidermal mRNA. Predicted amino acid sequences of LCP17 and LCP22 are homologous to cuticle proteins from other insect species, including Manduca sexta, Drosophila melanogaster and Locusta migratoria. This fact suggests that these cuticle protein genes originated from a common ancestral gene and have been conserved during evolution. Northern blot hybridization demonstrated that the expression of LCP17 as well as LCP22 mRNA is controlled in a stage-specific manner in the epidermis of the final instar larvae, suggesting a common regulatory mechanism for transcription of these two intermolt genes.

A case of dyshormonogenetic goiter complicated with growth hormone deficiency.

A 17-year-old male patient with dyshormonogenetic goiter complicated with follicular adenoma and growth hormone deficiency is described. He had short stature (-2.3 SD), diffuse goiter and a particularly large nodule in the right lobe of the thyroid gland. The endocrinological studies revealed slight hypothyroidism. He underwent surgical removal of the tumor, diagnosed histopathologically as follicular adenoma, embryonal type; and the diffuse goiter was diagnosed as dyshormonogenetic goiter. It was speculated that long-term hypersecretion of thyroid stimulating hormone (TSH) caused adenoma in the thyroid gland. Although the goiter disappeared after oral thyroxine replacement therapy, his height gain remained poor. Then, he was diagnosed partial growth hormone deficiency, and growth hormone therapy improved his height gain. It has been reported that a high percentage of patients with congenital hypothyroidism have additional anomalies. This is, however, the first reported case of dyshormonogenetic goiter complicated with growth hormone deficiency.

The division abnormally delayed (dally) gene: a putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila.

We have devised a genetic screen to obtain mutants affecting cell division patterning in the developing central nervous system of Drosophila. The division abnormally delayed (dally) locus was identified using a combination of "enhancer trap" and behavioral screening methods. The ordered cell cycle progression of lamina precursor cells, which generate synaptic target neurons for photoreceptors, is disrupted in dally mutants. The first of two lamina precursor cell divisions shows a delayed entry into mitosis. The second division, one that is triggered by an intercellular signal from photoreceptor axons, fails to take place. Similar to lamina precursors, cells that generate the ommatidia of the adult eye show two synchronized divisions found along the morphogenetic furrow in the eye disc and the first division cycle in dally mutants displays a delayed progression into M phase like that found in the first lamina precursor cell division. dally mutations also affect viability and produce morphological defects in several adult tissues, including the eye, antenna, wing and genitalia. Sequencing of a dally cDNA reveals a potential open reading frame of 626 amino acids with homology to a family of Glypican-related integral membrane proteoglycans. These heparan sulfate-containing proteins are attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol linkage. Heparan sulfate proteoglycans may serve as co-receptors for a variety of secreted proteins including fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor and members of the Wnt, TGF-beta and Hedgehog families. The cell division defects found in dally mutants implicate the Glypican group of integral membrane proteoglycans in the control of cell division during development.

[Serum ECP level of infants of atopic dermatitis].

Forty-seven infants with atopic dermatitis (AD) were examined for ECP (eosinophil cationic protein) levels in sera at 3 to 4, 6 to 7, 12 and 18 months after birth. ECP levels in AD were significantly higher than those of control infants at every examination. The levels of ECP in AD were significantly correlated with the severity of dermatitis judged by a slightly modified version of the method of Businco et al. We concluded that ECP levels in sera are a useful marker for the severity of dermatitis in infants with AD.

Structure and developmental expression of a larval cuticle protein gene of the silkworm, Bombyx mori.

Structure and expression of the gene for a larval cuticle protein of the silkworm, Bombyx mori were studied. A major cuticle protein, termed 'LCP30' was purified from the urea extract of integuments of the fifth (final) instar larvae. Immunoblot analysis by use of the anti-LCP30 antibody revealed that LCP30 begins to accumulate in larvae as early as 10 h after hatch and is present throughout the larval stages. The LCP30 epitope is also detectable in the adult abdominal integument but is absent from pupal integument and adult wing. Screening of an epidermal cDNA expression library with the antibody probe yielded a cDNA clone for LCP30. Primary structure deduced from the cDNA sequence showed that LCP30 bears an arginine-glycine-aspartate (RGD) sequence. The region around this domain exhibits striking similarity with the amino acid sequences found in vertebrate collagens. The genomic DNA clone coding for LCP30 was isolated by screening a B. mori gene library with the LCP30 cDNA probe. The gene consists of five exons interspersed by four introns spanning over 2.7 kb region of chromosomal DNA. The LCP30 mRNA is detectable at high levels at larval intermolt stages, gradually declines after the fourth molt and totally disappears at mid-fifth larval instar, indicating that the expression of LCP30 gene is regulated in a stage-specific fashion in the epidermal cells. Topical application of a juvenile hormone analogue (methoprene) to the fifth instar larvae followed by RNA blot and S1 nuclease mapping analyses of the epidermal RNA proved that juvenile hormone activates transcription of the LCP30 gene.

[Clinical study on S-1108 in treatment of pediatric infections].

To evaluate the efficacy, the safety and the usefulness of a novel and esterified cephem antibiotic for oral use, S-1108, in pediatric infections, a clinical trial was performed. The study subjects were 15 patients including 9 with acute pharyngitis, 1 with acute tonsillitis, 2 with acute bronchitis, 1 with chronic pyelonephritis, 1 with acute abscess of the skin and 1 with impetigo contagiosa. S-1108 was administered orally at a dose of 3.7 mg/kg to 12.5 mg/kg t.i.d. for 4 to 9 days. Clinical effects were excellent in 7 cases, good in 6, fair in 1 and poor in 1. The Overall efficacy rate was 86.7%. Bacteriologically, causative organisms were all eradicated in evaluable 4 cases. As to side effects, diarrhea was observed in 2 cases. No abnormal laboratory test values were obtained.