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Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats.

The objective of this study was to determine if levels of repressors to myogenic regulatory factors (MRFs) differ between muscles from young adult and aged animals. Total RNA from plantaris, gastrocnemius, and soleus muscles of Fischer 344 x Brown Norway rats aged 9 mo (young adult, n = 10) and 37 mo (aged, n = 10) was reverse transcribed and then amplified by PCR. To obtain a semiquantitative measure of the mRNA levels, PCR signals were normalized to cyclophilin or 18S signals from the corresponding reverse transcription product. Normalization to cyclophilin and 18S gave similar results. The mRNA levels of MyoD and myogenin were approximately 275-650% (P < 0.001) and approximately 500-1,100% (P < 0.001) greater, respectively, in muscles from aged compared with young adults. In contrast, the protein levels were lower in plantaris and gastrocnemius muscles and similar in the soleus muscle of aged vs. young adult rats. Id repressor mRNA levels were approximately 300-900% greater in fast and slow muscles of aged animals (P < or = 0.02), and Mist 1 mRNA was approximately 50% greater in the plantaris and gastrocnemius muscles (P < 0.01). The mRNA level of Twist mRNA was not significantly affected by aging. Id-1, Id-2, and Id-3 protein levels were approximately 17-740% greater (P < 0.05) in hindlimb muscles of aged rats compared with young adult rats. The elevated levels of Id mRNA and protein suggest that MRF repressors may play a role in gene regulation of fast and slow muscles in aged rats.

Aging↗

Id genes in nervous system development.

Id genes encode helix-loop-helix proteins that function to mediate processes important for normal development including cellular differentiation, proliferation and apoptosis. Id proteins act as negative regulators of other transcription factors, which are essential for cell determination and differentiation in diverse cell types, and interact with proteins important for cell cycle regulation. Studies of Id gene expression in the nervous system and in neural cells in culture indicate that Id proteins contribute to the regulation of mammalian nervous system development. Also, recognition of a wide variety of proteins with which Id transcription factors are capable of interacting suggests that it will be possible to understand more precisely their specific functions and importantly how these are integrated.

Amino Acid Sequence↗

Id proteins--tumor markers or oncogenes?

The Id (Inhibitor of differentiation or Inhibitor of DNA-binding) proteins act as dominant negative inhibitors of differentiation-specific basic Helix-Loop-Helix (bHLH) transcription factors. Id proteins negatively regulate cellular differentiation and they induce proliferation by modulating different cell cycle regulators both by direct and indirect mechanisms. Ectopic expression of Id proteins in tissue culture models can result in cellular immortalization and abrogation of differentiation processes. Recent reports show that Id proteins are overexpressed in various cancer types implying a role of these regulatory proteins in carcinogenesis. This review focuses on the biology of the Id proteins and their role as potential oncogenes.

Amino Acid Sequence↗

Identification and genetic analysis of Panama-genotype Venezuelan equine encephalitis virus subtype ID in Peru.

Venezuelan equine encephalitis (VEE) virus was isolated in 1993, 1994, and 1995 from human cases of acute, undifferentiated, febrile illness in the Peruvian Amazon Basin. Two virus isolates were recovered in 1994 from Peruvian soldiers at a jungle outpost near Pantoja in northern Peru, and 10 isolates were obtained from military personnel and civilians in 1993-1995 in Iquitos, an urban center in northeastern Peru. The genetic relationship of these isolates to other VEE virus strains was determined by sequencing 856-867 nucleotide reverse transcription-polymerase chain reaction fragments derived from the PE2 glycoprotein gene. The sequences were compared with those of other VEE virus strains, including representatives of the IAB, IC, ID, IE, II, and IIIC subtypes. The two Pantoja isolates were most closely related to subtype IC and ID viruses previously isolated in Colombia and Venezuela, and to the ID viruses isolated during the 1970s in Iquitos. All of the recent Iquitos isolates were similar to one another, but they were more closely related to Panamanian ID strains than to isolates previously obtained in Iquitos, Peru, or in Colombia and Venezuela. The recent Iquitos VEE viral isolates were the first Panama-genotype VEE ID virus strains identified outside of the Republic of Panama.

Animals↗

Identification of differentially expressed genes in esophageal squamous cell carcinoma (ESCC) by cDNA expression array: overexpression of Fra-1, Neogenin, Id-1, and CDC25B genes in ESCC.

PURPOSE: This study aims to identify differentially expressed genes in esophageal squamous cell carcinoma (ESCC) through the use of a membrane-based cDNA array. EXPERIMENTAL DESIGN: Two newly established human ESCC cell lines (HKESC-1 and HKESC-2) and one corresponding to a morphologically normal, esophageal epithelium tissue specimen, prospectively collected from the HKESC-2-related patient, were screened in parallel using a cDNA expression array containing gene-specific fragments for 588 human genes spotted onto nylon membranes. RESULTS: The results of cDNA expression array showed that 53 genes were up-regulated 2-fold or higher and 8 genes were down-regulated 2-fold or higher in both ESCC cell lines at the mRNA level. Semiquantitative RT-PCR analysis of a subset of these differentially expressed genes gave results consistent with cDNA array findings. Four of the differentially expressed genes that belong to the categories of oncogenes/tumor suppressor genes (Fra-1 and Neogenin) and cell cycle-related genes (Id-1 and CDC25B) were studied more extensively for their protein expression by immunohistochemistry. The two ESCC cell lines and their corresponding primary tissues, 61 primary ESCC resected specimens and 16 matching, morphologically normal, esophageal epithelium tissues were analyzed. The immunostaining results showed that Fra-1, Neogenin, Id-1, and CDC25B were overexpressed in both ESCC cell lines and their corresponding primary tumors at the protein level, validating the microarray findings. The results of the clinical specimens showed that the Fra-1 gene was overexpressed in ESCC compared with normal esophageal epithelium in 53 of 61 cases (87%), Neogenin in 57 of 61 cases (93%), Id-1 in 57 of 61 cases (93%), and CDC25B in 48 of 61 cases (79%). Furthermore, the expression of Fra-1, Neogenin, and Id-1 in ESCC correlated with tumor differentiation. CONCLUSIONS: Overall, this study demonstrates that multiple genes are differentially expressed in ESCC and provides the first evidence that oncogenes Fra-1 and Neogenin and cell cycle-related genes Id-1 and CDC25B are overexpressed in ESCC.

Carcinoma, Squamous Cell↗

Transcriptional regulation in cardiac muscle. Coordinate expression of Id with a neonatal phenotype during development and following a hypertrophic stimulus in adult rat ventricular myocytes in vitro.

The transcriptional regulatory mechanisms in heart muscle that direct cardiac development and allow for a flexible, adaptive response to physiologic stress are not well understood. We demonstrate that a negative regulator of gene transcription termed Id that has been described predominantly in proliferating cell lines and in undifferentiated tissue during growth, is expressed in freshly isolated terminally differentiated adult rat ventricular myocytes, in contrast to most other tissues in the adult rat. Id mRNA expression is regulated in ventricular myocytes during post-natal development, peaking at the transition from hyperplastic to hypertrophic growth at day 17 in the rat, declining subsequently to lower, stable levels in adult myocytes. Although Id mRNA becomes undetectable in adult ventricular myocytes 48 h following isolation in the absence of serum, it can be rapidly reinduced by an alpha-adrenergic agonist, accompanied by increased protein synthesis and the reexpression, in defined media, of the neonatal genes prepro-ANP and skeletal muscle alpha-actin. Thus, the differential regulation of Id during cardiac development, the presence of Id mRNA in normal cardiac myocytes, and its increased expression following a hypertrophic stimulus all suggest a role for this transcriptional regulator in the control of cardiac muscle cell phenotype.

Aging↗

Overexpression of Id-1 in gastric adenocarcinoma: implication for a novel diagnostic marker.

Gastric cancer is the second most common cause of cancer-related death worldwide and the highest incidence of this cancer has been reported in Asia, especially in China. Identification of early stage lesions is vital in achieving high survival rate. However, due to the lack of reliable biomarkers, the majority of gastric cancer is presented at an advanced stage. Recently, it has been reported that Id-1, a helix-loop-helix protein, may be a valuable diagnostic marker in many types of human cancer. In this study, we evaluated Id-1 protein expression in gastric cancer specimens and compared it with non-malignant tissues. In addition, to investigate whether Id-1 expression levels were associated with the aggressiveness of this disease as implicated in other cancer types, we also assessed Id-1 expression levels in primary tumours and their lymph node metastasized lesions. Our results indicate that up-regulation of Id-1 is a frequent event in gastric cancer but its expression levels are not associated with tumour metastasis. Our evidence provides a possible novel marker for the diagnosis of gastric cancer.

Adenocarcinoma↗

Expression of Id proteins in human hepatocellular carcinoma: relevance to tumor dedifferentiation.

Several studies reported that Id (Inhibitor of DNA binding or Differentiation) proteins, helix-loop-helix transcription factors, have important roles in differentiation, cell cycle and angiogenesis in various cells. However, the role of Id proteins in hepatocellular carcinoma (HCC) remains unclear. We examined the immunohistochemical expression of Id1, Id2 and Id3 proteins in 54 surgically resected HCCs with surrounding HCV or HBV-related chronic hepatitis (n=30) and liver cirrhosis (n=24). All non-cancerous livers exhibited immunoreactivity for Id proteins and the expression increased from chronic hepatitis to cirrhosis. In HCCs (n=45), well-differentiated tumors mostly exhibited strong or moderate immunostaining for all Id proteins, while proportion of the samples with weak or no expression increased with tumor dedifferentiation and frequently observed in poorly (66.7, 93.3 and 93.3% respectively for Id1, 2, 3) or undifferentiated (100% for all Ids) HCCs. Clinicopathological survey demonstrated a significant correlation between Id1, 2 and 3 expression and differentiation of carcinoma (p=0.0044, 0.0014 and 0.0014, respectively) although univariate analysis indicated that high expression of Id1 was significant predictive factor for longer disease-free survival of the patients (p=0.047). A similar tendency was also observed with Id2 and Id3. The present study demonstrate high expression of Id1, 2 and 3 in well-differentiated HCC and low expression in advanced dedifferentiated HCC, in contrast to its continuous expression during breast, prostate and colon carcinogenesis. These findings suggested that Id1, 2 and 3 might play a role in the early stages of hepatocarcinogenesis, but not in the development of advanced carcinoma, and might consequently be related to HCC dedifferentiation.

Blotting, Western↗

ID sequences in the genes of three brain-specific proteins.

We characterized the brain-specific gene coding for rat S-100 protein beta-subunit and found three "brain identifier (ID)" elements, which have been proposed to regulate the gene expression in rat brain. The nucleotide sequences of these elements corresponded well with that of the consensus ID element and were clearly different from those of "ID-like" elements in rat beta B1-crystallin gene, etc. ID elements were also observed in the flanking regions of rat neuron-specific enolase and cholecystokinin genes, which were expressed in the neuronal cells. Direct repeats were observed in the regions flanking ID elements.

Animals↗

An evaluation of a new gel system (ID-gel) for antibody screening and identification.

An evaluation of the new "ID-Gel System" (Diamed) for the detection of red cell antibody-antigen reactions was undertaken in the King Khalid University Hospital Blood Bank. Antibody screening was performed in 700 patient samples using both the conventional test tube (bovine albumin and LISS) techniques and the new ID-gel technique. The results obtained were as follows: a) 70 antibodies of various types were detected by the bovine albumin technique. b) 98 antibodies were spotted by LISS. c) The ID-gel system detected 122 antibodies (17.43%) which included all those detected by the other two techniques. It was concluded that the ID-gel system is a far more sensitive technique than the conventional Blood Bank immuno-haematological tests. In addition, the ID-system has the further advantage of standardization of the test, shorter incubation time, no washing procedure and reduces the technologist time, effort and perhaps number.

Blood Grouping and Crossmatching↗

[Glucocorticoids specifically regulate the expression of dispersed repeating sequences in liver cells. Detection of small ID-like antisense RNA].

Differential regulation of the expression of various families of the rat genome interspersed repetitive sequences by glucocorticoid hormones in liver has been demonstrated. Small ID-like RNA complementarily associated with high-molecular-weight cytoplasmic poly(A)-containing RNAs has been identified. The results obtained indicate that glucocorticoids hormones induce expression of the B2, ID, and L1 families in rat liver. The content of the transcripts of these repetitive elements is increased by the hormones in the molecules of heterogeneous nuclear RNA. In the molecules of high-molecular-mass poly(A)-containing cytoplasmic RNA glucocorticoids increase the content of the ID-homologous sequences. In the population of the small specific RNA the content of the ID-homologous transcripts is also increased. A suggestion is put forward about the involvement of the ID-like element in the hormonal posttranscriptional regulation of gene expression at the mRNA level.

Animals↗

The helix-loop-helix protein Id inhibits differentiation of murine erythroleukemia cells.

Id is considered to be a negative regulator of basic helix-loop-helix proteins, which play important roles in cell type-specific transcription and cell lineage commitment. The Id gene was first cloned in murine erythroleukemia (MEL) cells, which can be induced to differentiate toward erythrocytes with Me2SO, and its mRNA decreases after differentiation in various types of cells. In this report, we demonstrate that overexpression of Id interferes with MEL cell differentiation and that inhibition of differentiation is accompanied by reduction in expression of three erythroid-specific genes. While down-regulation of Id is an early event in the differentiation process of MEL cells, E-box binding activity of these cells increases only at a later stage of differentiation, and this late increase is reduced by the overexpression of Id in the early stage. Sequential changes in the activity of several basic helix-loop-helix proteins thus appeared to be involved in erythroid differentiation.

Animals↗

A polymorphism of the X-linked gene IDS increases the number of females informative for transcriptional clonality assays.

Studies of clonality have been essential for understanding the hierarchy of hematopoiesis and the biology of malignancies. Most clonality assays are based on the X chromosome inactivation phenomenon in females; these assays detect protein polymorphisms, differences in DNA methylation, or transcripts of the active X chromosome. Assays based on protein polymorphisms or DNA methylation have significant shortcomings. The major disadvantage of transcriptional assays is their limited applicability since only approximately half of females are informative for these studies. We have developed a new transcriptional assay based on an exonic polymorphism of the X-chromosome gene IDS. This gene is located in the same X-chromosome region (Xq28) as G6PD and p55, two genes with exonic polymorphisms for which we previously developed transcriptional assays. We developed non-radioactive PCR-based assays for rapid screening of genotype and determination of clonality. We also report reaction conditions for a quantitative ligase detection assay of IDS allelic transcripts. The frequency of the IDS polymorphism is 46% in Caucasian females and 39% in African-American females; in combination with G6PD and p55, 76% of Caucasian females and 62% of African-American females are informative for these assays. While this gene is highly polymorphic in Caucasian and African-American females, it is not informative in Oriental females. We established that the IDS gene is in linkage equilibrium with G6PD and p55. Unlike methylation-based assays, this assay is suitable for studying clonality in non-nucleated cells such as platelets and reticulocytes. With the discovery of exonic polymorphisms of other X-chromosome genes, all females should eventually be suitable for X-chromosome transcriptional clonality analysis.

Antigens, Bacterial↗

Effects of the sabal serrulata extract IDS 89 and its subfractions on 5 alpha-reductase activity in human benign prostatic hyperplasia.

Extract from fruit of Sabal serrulata are used in the treatment of human benign prostate hyperplasia (BPH). Therefore, it is of interest whether this phytopharmacon has any influence on the androgen metabolism in the human prostate. It was found that the extract IDS 89 of Sabal serrulata inhibited dose dependently 5 alpha-reductase activity in the epithelium and stroma of human BPH, the mean inhibition being 29% and 45%, respectively. This inhibitory effect is mainly due to the saponifiable subfraction of IDS 89 showing a mean 5 alpha-reductase inhibition of 39% and 38% in epithelium and stroma, respectively. The inhibition was dose dependent and noncompetitive. At a testosterone concentration of 580 nM as substrate for 5 alpha-reductase, the main fatty acids of the extract IDS 89 gave rise to a percentual enzyme inhibition in the epithelium and stroma as follows: 51% and 42% (lauric acid), 5% and 0% (oleic acid), 43% and 34% (myristic acid), 2% and 0% (palmitic acid), respectively. The inhibitory effect of lauric acid was noncompetitive and dose dependent up to a concentration of 0.2 nM, the maximal inhibition in the epithelium and stroma being 52% and 45%, respectively. The nonsaponifiable subfraction, consisting mainly of phytosterols, showed a mean inhibition of 5 alpha-reductase in the epithelium and stroma of 15% and 10%, respectively. Finally, the hydrophilic subfraction, containing carbohydrates, amino acids, and polysaccharides, showed no inhibitory effect. The present in vitro studies suggest that the Sabal serrulata extract IDS 89 has an inhibitory effect on 5 alpha-reductase in the epithelium and stroma of human BPH. This inhibition is mainly due to the fatty acids of the saponifiable subfraction.

Cholestenone 5 alpha-Reductase↗

Synthesis and conformational properties of protein fragments based on the Id family of DNA-binding and cell-differentiation inhibitors.

Id proteins are dominant negative regulators of the helix-loop-helix (HLH) transcription factors and are important during development, especially by preventing cell differentiation while inducing cell proliferation. In contrast, they are poorly expressed in healthy adults but are found in several tumor types. The Id HLH motif is responsible for the inhibitory activity, whereas not much is known about the role of the N- and C-termini. In the presented work, synthetic peptides reproducing the HLH, the N-terminal region, and the C-terminal region of the Id proteins were characterized by CD. The four HLH sequences built highly stable helical conformations, whereas the N- and C-termini were unstructured, with the exception of an alanine-rich fragment preceding the Id4 HLH motif. Deletion of the loop connecting the two helices led to helix destabilization for all four Id HLH peptides. In addition, modifications of the amino acid composition within the hydrophobic face of the helices of the Id1 HLH peptide induced conformational changes, mostly associated with loss of helix content. Moreover, a fragment containing the helix-2 and the C-terminus of the Id1 protein did not show any helical character. Therefore, both the helix propensity and stability of the HLH domain were shown to be strongly dependent on favorable interhelical contacts. In contrast, it is suggested that the regions beyond this domain could rather play a destabilizing role, for example, by increasing the flexibility of the folded protein.

Amino Acid Motifs↗

Sequence of the human iduronate 2-sulfatase (IDS) gene.

Deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13) results in the storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, which leads to the lysosomal storage disorder mucopolysaccharidosis type II. Three overlapping genomic clones derived from an X-chromosome-specific library containing the entire IDS gene were isolated and the sequences of the intron boundaries and the 5' promoter region were determined. The IDS gene is split into nine exons spanning approximately 24 kb. The potential promoter for IDS lacks a TATA box but contains GC box consensus sequences, consistent with its role as a housekeeping gene. A polypyrimidine-like repeat is found in intron 1.

Base Sequence↗

Identification of an alternative transcript from the human iduronate-2-sulfatase (IDS) gene.

Iduronate-2-sulfatase (IDS) is involved in the degradation of heparan sulfate and dermatan sulfate in the lysosomes, and a deficiency in this enzyme results in Hunter syndrome. A 2.3-kb cDNA clone that contains the entire coding sequence of IDS has previously been reported. Here we describe the identification of a 1.4-kb transcript that may encode an IDS-like enzyme. The predicted protein is identical to the previously described enzyme, except for the absence of the 207-amino-acid COOH-terminal domain, which is replaced by 7 amino-acids. Our data suggest that there might exist an additional form of the IDS enzyme in humans. The results from this study may have implications for the pathogenesis of the Hunter syndrome.

Alternative Splicing↗

Reciprocal Id expression and myelin gene regulation in Schwann cells.

Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.

Aging↗