Inhibition of IGFBP-5 binding to extracellular matrix and IGF-I–stimulated DNA synthesis by a peptide fragment of IGFBP-5

Insulin-like growth factor binding protein-5 (IGFBP-5) is synthesized and secreted by smooth muscle cells (SMC). IGFBP-5 synthesis is stimulated five- to sixfold by IGF-I, and IGFBP-5 has been shown to augment IGF-I–stimulated DNA synthesis in this cell type. The ability of IGFBP-5 to augment the SMC response to IGF-I is dependent upon its binding to extracellular matrix. A highly charged region of IGFBP-5 that contains amino acids in positions 201–218 has been shown to mediate binding of IGFBP-5 to human fibroblast extracellular matrix (ECM), and a synthetic peptide containing this sequence inhibits IGFBP-5 binding to fibroblast ECM. In this study we show that exposure of SMC cultures that are constituitively synthesizing IGFBP-5 to a synthetic peptide (termed peptide A) containing this sequence has no effect on its synthesis but reduces its abundance within the ECM. The addition of increasing concentrations of the peptide to SMC cultures resulted in a concentration-dependent reduction in ECM-associated IGFBP-5. In contrast, a control peptide (peptide B), which contained the region of amino acids in positions 131–141 and had a similar charge-to-mass ratio, caused a minimal decrease in ECM binding. This effect was functionally significant since the addition of 10 μg/ ml of peptide A inhibited the cellular replication response to 10 ng/ ml IGF-I by 51%, and peptide B had no effect. The effects of peptide A were not due to nonspecific cytotoxicity since it had no inhibitory effect on the response of these cells to human serum and was associated with only minimal inhibition of the cellular response to platelet-derived growth factor. The findings suggest that inhibiting IGFBP-5 binding to porcine SMC ECM results in reduced cellular responses to IGF-I. J. Cell. Biochem. 71:375–381, 1998. © 1998 Wiley-Liss, Inc.     

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.     

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.     

A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

Differential expression of insulin-like growth factor-I and insulin-like growth factor binding protein-1 in the diabetic rat

Summary Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10–30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p < 0.05) and diaphragm (p < 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p < 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-1 by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.     

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.     

Insulin-like growth factor binding protein 5 and type-1 insulin-like growth factor receptor are differentially regulated during apoptosis in cerebellar granule cells

Neuronal apoptosis is considered to play a significant role in several neuropathological conditions. However, the molecular mechanisms underlying neuronal apoptosis are poorly understood. Insulin-like growth factor (IGF) signalling is considered to be an important regulator of neuronal differentiation, survival and apoptosis. We have examined the expression of two members of the IGF system, insulin-like growth factor binding protein 5 (IGFBP-5) and the type-1 IGF receptor (IGF1R), during apoptosis of rat cerebellar granule cells (CGCs) in vitro. We describe a prominent downregulation of IGFBP-5 mRNA and protein expression. We also show that IGF-I increases IGFBP-5 expression in CGCs and that the downregulation of IGFBP-5 mRNA can be suppressed by inhibiting mRNA synthesis with actinomycin D. The expression of IGF1R mRNA showed a transient upregulation during potassium chloride (KCl) deprivation induced apoptosis, in contrast to the IGF1R protein level, which was downregulated during KCl deprivation. Our results provide insight into the expression of IGF-related genes during neuronal apoptosis, and indicate that they mediate a protective response to the withdrawal of trophic stimulation. It seems that the expression of IGFBP-5 and IGF1R is regulated to maximize the availability of IGF and the activity of IGF-triggered survival signalling.     

Structure and expression of a chicken insulin-like growth factor I precursor

Insulin-like growth factor I (IGF-I) is a 70 amino acid growth-promoting polypeptide whose sequence and functions have been highly conserved among mammals. As an initial step in defining the role of IGF-I in other vertebrate species, we have isolated and characterized an IGF-I cDNA from the chicken. This cDNA encodes a 153 amino acid primary translation product which resembles in structure and sequence the IGF-IA protein of mammals. There is strong amino acid conservation between chicken and mammalian IGF-I throughout the entire protein. Sixty of 70 amino acids are identical in mature IGF-I among the chicken, rat, and human peptides, with five differences being localized to the C domain, and two to the D region. A comparable degree of amino acid identity is found in the COOH-terminal extension peptide (28/35 residues). At the NH2-terminus, where there is more amino acid divergence (32/48 identities), the most 5'-AUG codon is the only methionine residue conserved among all three species, suggesting that it functions as the authentic translation initiation site, an observation supported by cell-free studies of biosynthesis and cotranslational proteolytic processing. The pattern of IGF-I gene expression appears to be simpler in chickens than in mammals, since a single predominant mRNA of 2.6 kilobases can be detected in liver polyadenylated RNA on Northern blots. In the chicken, as in rats and humans, IGF-I mRNA is synthesized in multiple tissues, including liver, brain, skeletal muscle, and heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).     

Expression of the congenital heart disease 5/tryptophan rich basic protein homologue gene during heart development in Medaka fish,Oryzias latipes

The congenital heart disease 5 (CHD5)/tryptophan rich basic protein (WRB) is a protein containing a tryptophan‐rich carboxy‐terminal region, which was discovered in the human fetal heart. In humans, this CHD5/WRB is located between the markers ACTL5‐D21S268 within the Down syndrome (DS) Region‐2 at chromosome 21. Congenital heart disease is commonly linked to DS patients. The functions of this gene product are unknown. To identify the functions of CHD5/WRB in heart formation during embryogenesis, the medaka CHD5 cDNA (mCHD5) was isolated and its gene expression pattern and the localization of its gene product were investigated. The obtained mCHD5 belongs to the CHD5 superfamily, whose members include coiled‐coil proteins. The mCHD5 gene was found to be expressed in the developing heart after stage 28 at which the chamber (ventricle and atrium) differentiation in the heart tube is initiated in the embryo. Its gene product was also detected in the developing heart at embryonic stage 28 and 35. Knocking‐down of mCHD5 function caused severe cardiac disorder, including abnormal chamber differentiation, abnormal looping and ocular abnormality such as Cyclops. Our results provide the mCHD5 gene expression pattern as well as its physiological role during heart formation in a vertebrate model system.     

A common myosin light chain is expressed in chicken embryonic skeletal, cardiac, and smooth muscles and in brain continuously from embryo to adult

We have isolated cDNA clones of the mRNA for chick embryonic myosin light chain (MLC), L23, by cross-hybridization with chicken skeletal muscle MLC1 cDNA. The identification of the isolated cDNAs was carried out by in vitro translation of hybrid-selected mRNA. Sequence analysis of the cloned cDNAs revealed that the cDNA insert contained 832 nucleotides and predicted a polypeptide of 185 amino acids with a calculated molecular weight of 20,687. The deduced amino acid sequence for L23 showed high sequence similarities to those of adult alkali type MLCs from various tissues, indicating that L23 belongs to the alkali MLC group. Using the cloned cDNA as a hybridization probe, we have revealed by RNA blot analysis that the expression of L23 mRNA was regulated in temporal and tissue-specific manners. The L23 mRNA of 1.1 kilobases is transiently expressed in embryonic skeletal, cardiac, and smooth muscles of chickens. It is also found in the brain of chickens during all stages of development so far investigated. Only a single gene for L23 was detected by Southern blot of chick genomic DNA. We therefore suggest that L23 is expressed from a single gene in both embryonic muscles and brain.     

虾夷扇贝脂多糖诱导的肿瘤坏死因子LITAF基因的克隆及表达分析

脂多糖诱导的肿瘤坏死因子(Lipopolysaccharide-induced TNF-alpha factor,LITAF)是一类重要的炎症细胞因子,在先天性免疫系统中发挥重要的介质作用。文章根据虾夷扇贝LITAF基因EST序列,应用RACE技术克隆了虾夷扇贝LITAF全长cDNA,对序列及编码的氨基酸进行生物信息学分析。结果显示,该基因cDNA全长1 551 bp,其5′非编码区包含76 bp,3′非编码区包含1 001 bp;开放阅读框(ORF)为474 bp,编码157个氨基酸,氨基酸序列中存在一个保守的LITAF结构域;理论分子量16.99 kDa,等电点为6.24。LITAF基因序列为3 698 bp,由3个外显子和两个内含子组成。利用实时荧光定量PCR技术分析LITAF在虾夷扇贝不同组织、不同胚胎发育阶段以及鳗弧菌(Vibrio anguillarum)刺激后各时间段的表达情况。结果表明:LITAF基因在所检测的6个成体组织中均有表达,其中肾脏的表达量最高;胚胎发育的7个时期中,担轮幼体时期表达量最高;菌刺激36 h实验组与对照组的表达量差异大。LITAF基因是LITAF家族的一员,推测LITAF基因参与虾夷扇贝的先天性免疫反应。