Search PubMed⌕ Search

Biomedical subjects

S I Tomarev

Publications and source records attributed to S I Tomarev.

At least 19 recordsLinked to original sources
PubMed2004

The homeobox transcription factor Prox1 is highly conserved in embryonic hepatoblasts and in adult and transformed hepatocytes, but is absent from bile duct epithelium.

Prox1 is a transcription factor with two highly conserved domains, a homeobox and a prospero domain. It has been shown that Prox1 knock-out mice die during early embryonic stages and display a rudimentary liver. We have studied the expression of Prox1 at RNA and protein levels in chick, rat, mouse and human liver and in transformed and non-transformed hepatic cell lines. Prox1 is expressed in early embryonic hepatoblasts and is still expressed in adult hepatocytes. Prox1 protein is located in the nuclei of hepatoblasts, which grow into the neighboring embryonic mesenchyme. The expression pattern in chick, mouse, rat and human embryos is highly conserved. Besides albumin and alpha-fetal protein, Prox1 belongs to the earliest markers of the developing liver. In adult liver, Prox1 is expressed in hepatocytes but is absent from bile duct epithelial and non-parenchymal cells (Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells and myofibroblasts). Isolated primary hepatocytes and hepatoma cell lines (HepG2, Hep3B) are Prox1 positive, whereas the immortalized murine liver cell-line MMH, which constitutively expresses the receptor c-met, is Prox1 negative. Transfection of MMH with Prox1 cDNA increases the expression level significantly as compared to control transfectants. In HepG2 and Hep3B, the Prox1 levels are even up to 100 times higher. Our studies show that Prox1 is a highly conserved transcription factor, expressed in hepatocytes from the earliest stages of development into adulthood and over-expressed in hepatoma cell lines. Its absence from bile duct epithelial cells suggests a function for the specification of hepatoblasts into hepatocytes. The genes controlled by Prox1 need to be studied in the future.

Animals↗
PubMed2001

LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis.

Lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 is thought to be restricted to lymph vessels and has been used as such to show that tumor lymphangiogenesis occurs on overexpression of lymphangiogenic factors in mouse tumor models. However, these studies have not yet been corroborated in human tumors. Here we show, first, that LYVE-1 is not exclusive to the lymph vessels. Indeed, LYVE-1 is also present in normal hepatic blood sinusoidal endothelial cells in mice and humans. Surprisingly, LYVE-1 is absent from the angiogenic blood vessels of human liver tumors and only weakly present in the microcirculation of regenerative hepatic nodules in cirrhosis, though both vessels are largely derived from the liver sinusoids. Second, we propose a novel approach to identify lymphatics in human and murine liver. By combining LYVE-1 and Prox 1 (a transcription factor) immunohistochemistry, we demonstrate that lymphatics are abundant in cirrhosis. In contrast, in human hepatocellular carcinoma and liver metastases, they are restricted to the tumor margin and surrounding liver. The absence of intratumor lymphatics in hepatocellular carcinomas and liver metastases may impair molecular and cellular transport in these tumors. Finally, the presence of LYVE-1 in liver sinusoidal endothelia suggests that LYVE-1 has functions beyond the lymph vascular system.

Animals↗
PubMed2001

Development of the avian lymphatic system.

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1.

Animals↗
PubMed2001

Intraocular pressure in genetically distinct mice: an update and strain survey.

BACKGROUND: Little is known about genetic factors affecting intraocular pressure (IOP) in mice and other mammals. The purpose of this study was to determine the IOPs of genetically distinct mouse strains, assess the effects of factors such as age, sex and time of day on IOP in specific strain backgrounds, and to assess the effects of specific candidate gene mutations on IOP. RESULTS: Based on over 30 studied mouse strains, average IOP ranges from approximately 10 to 20 mmHg. Gender does not typically affect IOP and aging results in an IOP decrease in some strains. Most tested strains exhibit a diurnal rhythm with IOP being the highest during the dark period of the day. Homozygosity for a null allele of the carbonic anhydrase II gene (Car2n) does not alter IOP while homozygosity for a mutation in the leptin receptor gene (Leprdb) that causes obesity and diabetes results in increased IOP. Albino C57BL/6J mice homozygous for a tyrosinase mutation (Tyrc-2J) have higher IOPs than their pigmented counterparts. CONCLUSIONS: Genetically distinct mouse strains housed in the same environment have a broad range of IOPs. These IOP differences are likely due to interstrain genetic differences that create a powerful resource for studying the regulation of IOP. Age, time of day, obesity and diabetes have effects on mouse IOP similar to those in humans and other species. Mutations in two of the assessed candidate genes (Lepr and Tyr) result in increased IOP. These studies demonstrate that mice are a practical and powerful experimental system to study the genetics of IOP regulation and disease processes that raise IOP to harmful levels.

Age Factors↗
PubMed2001

Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.

Animals↗
PubMed2001

Prox1 is a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium and lymphangioblasts.

The lymphatic endothelium has mostly been thought to be derived by sprouting from specialized veins. Recently it has been shown that mice deficient for the homeobox transcription factor Prox1 are practically devoid of lymphatics. We have studied the expression of Prox1 mRNA and protein in chick embryos and human fetuses. In the chick, Prox1 is expressed in specific compartments of all germ layers. In the ectoderm, it is found in the neural tube, trigeminal, spinal and sympathetic ganglia and the retina, and also in placodal structures such as the lens, olfactory, otic, facial, glossopharyngeal and vagal placodes, and the apical ectodermal ridge. In the endoderm, Prox1 is a marker of hepatocytes, bile duct and pancreatic epithelium. In the mesoderm, weak expression is observed in cardiomyocytes, and strong expression in lymphatic endothelium. Identical expression domains are found in 19-week-old human fetuses. In day 6.5 chick embryos, there are several sites of contact of lymphatics with the jugular vein, which has a mixed endothelium of Prox1-positive and -negative cells. The only non-lymphatic endothelial cells expressing Prox1 are found on the concave side of the cardiac valves. To further analyse development of lymphatics, we studied early chick embryos and observed scattered Prox1-positive cells in the dermatome, giving rise to Prox1-positive lymphatic networks during subsequent development. Furthermore, the anlagen of the posterior lymph sacs and the paired thoracic duct can already be observed in day-4 chick embryos. Our studies show that lymphatics develop much earlier than previously described, and they mostly do not seem to be derived by sprouting from veins. In contrast, lymphangioblasts are present in the deep and superficial compartments of the early mesoderm, independently giving rise to the deep and superficial lymphatics.

Animals↗
PubMed2001

Targeted Disruption of the Myocilin Gene (Myoc) Suggests that Human Glaucoma-Causing Mutations Are Gain of Function.

Glaucoma is a heterogeneous eye disease and a major cause of blindness worldwide. Recently, primary open angle glaucoma (POAG)-associated mutations have been found in the trabecular meshwork inducible glucocorticoid response gene (TIGR), also known as the myocilin gene (MYOC), at the GLC1A locus on chromosome 1q21-q31. These mutations occurred in a subset of patients with juvenile- and adult-onset POAG and exhibited autosomal dominant inheritance. Ocular expression and its involvement in POAG suggest that TIGR/MYOC may have a role(s) in regulating intraocular pressure (IOP). Here, we report the generation and analysis of mice heterozygous and homozygous for a targeted null mutation in Myoc. Our study shows that Myoc mutant mice are both viable and fertile. Our in vivo findings further demonstrate that Myoc is not required for normal IOP or normal ocular morphology. The lack of a discernable phenotype in both Myoc-heterozygous and Myoc-null mice suggests that haploinsufficiency is not a critical mechanism for POAG in individuals with mutations in MYOC. Instead, disease-causing mutations in humans likely act by gain of function.

Animals↗
PubMed2001

Changes in mRNA levels of the Myoc/Tigr gene in the rat eye after experimental elevation of intraocular pressure or optic nerve transection.

PURPOSE: To isolate the rat Myoc/Tigr gene and investigate changes in its expression pattern in normal eyes and in eyes with either pressure-induced optic nerve damage or optic nerve transection. METHODS: Expression pattern of the rat Myoc/Tigr gene was investigated by Northern blot hybridization. Optic nerve damage and death of ganglion cells in the retina were induced unilaterally, by injection of hypertonic saline solution, episcleral vein cauterization, or optic nerve transection. The levels of mRNA for Myoc/Tigr were compared between several tissues of the control and surgically altered eyes, by using semiquantitative RT-PCR, real-time PCR, and Northern blot analysis. RESULTS: The rat Myoc/Tigr gene is 10 kb long and contains three exons. Among the eye tissues analyzed, Myoc/Tigr mRNA was detected in the combined tissues of the eye angle, sclera, cornea, retina, and optic nerve head. With pressure-induced optic nerve degeneration, the level of Myoc/Tigr mRNA decreased in the retina and the combined tissues of the eye angle, but increased in the optic nerve head. After optic nerve transection, the level of Myoc/Tigr mRNA increased in the retina, but did not change in the combined tissues of the eye angle. CONCLUSIONS: The decreased level of Myoc/Tigr mRNA in the retina after induction of elevated intraocular pressure compared with that in the control retina cannot be explained by ganglion cell death alone. Differences in Myoc/Tigr mRNA levels in eye tissues after elevation of intraocular pressure or optic nerve transection may reflect the activation of different signaling pathways involved in regulation of this gene.

Animals↗
PubMed2000

Active interaction of human A375 melanoma cells with the lymphatics in vivo.

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.

Allantoin↗
PubMed1999

O-Crystallin, arginine kinase and ferritin from the octopus lens.

Three proteins have been identified in the eye lens of the octopus, Octopus dofleini. A 22 kDa protein comprising 3-5% of the soluble protein of the lens is 35-43% identical to a family of phosphatidylethanolamine-binding proteins of vertebrates. Other members of this family include the immunodominant antigen of the filarial parasite, Onchocerca volvulus, putative odorant-binding proteins of Drosophila and a protein with unknown function of Caenorhabditis elegans. We have called this protein O-crystallin on the basis of its abundance in the transparent lens. O-Crystallin mRNA was detected only in the lens. Two tryptic peptides of another octopus lens protein, less abundant than O-crystallin, showed 80% identity to arginine kinase of invertebrates, a relative of creatine kinase of vertebrates. Finally, ferritin cDNA was isolated as an abundant cDNA from the octopus lens library. Northern blots showed that ferritin mRNA is not lens-specific.

Amino Acid Sequence↗
PubMed1999

Regulation of Prox 1 during lens regeneration.

PURPOSE: To determine the expression pattern of Prox 1 during the process of lens regeneration in the urodele Notophthalmus viridescens. METHODS: Polymerase chain reaction was performed to amplify a partial newt Prox 1 sequence. In situ hybridization and immunodetection methods were used to detect the Prox 1 mRNA and the Prox 1 protein, respectively. RESULTS: Prox 1 mRNA was present in the retina and in the lens (in the epithelium and bow region) of the intact eye. Prox 1 protein was found to be predominantly present in the lens and dorsal iris of the intact eye, although some trace levels of Prox 1 protein were detected in the ventral iris as well. After lentectomy, expression of the mRNA was also pronounced in the dorsal dedifferentiating iris and the regenerating lens. The ventral iris also expressed Prox 1 but seemingly at lower levels. Although Prox 1 protein showed upregulation in the dorsal iris during the process of lens regeneration, trace levels were also detected in the ventral iris. In the retina, Prox 1 protein was distributed in horizontal cells of the inner nuclear layer, whereas the mRNA was expressed in all layers of the retina. CONCLUSIONS: Prox 1 was unevenly distributed in the intact cells of the newt iris, with significantly higher levels of Prox 1 protein present in the dorsal versus the ventral margin. This protein was differentially regulated during the process of lens regeneration, with obvious upregulation in the dorsal iris. Prox 1 is the first transcriptional factor to be shown to be regulated in the dorsal versus ventral iris during the process of lens regeneration.

Amino Acid Sequence↗
PubMed1999

Identification of the region in the N-terminal domain responsible for the cytoplasmic localization of Myoc/Tigr and its association with microtubules.

Mutations in the MYOC/TIGR gene are associated with juvenile open-angle glaucoma and in some cases may be involved in the formation of sporadic primary open-angle glaucoma in humans. To better understand the functions of the MYOC/TIGR protein, its intracellular distribution was investigated using green fluorescent protein (GFP) as a marker. The results indicated that the recombinant mouse and human Myoc/Tigr-GFP proteins are located in the cytoplasm of the transfected cells in which they colocalize with microtubules. Deletion analysis demonstrated that the N-terminal region (positions 1-124 and 15-138 in the mouse and human proteins, respectively) encoded by exon 1 is critical for the cytoplasmic localization of Myoc/Tigr. Most of the known mutations in the human MYOC/TIGR gene implicated in juvenile and sporadic primary open-angle glaucoma formation are located outside the region responsible for the cytoplasmic localization of the protein. However, some of these mutations may alter the tertiary structure of the protein and subsequently modify its interaction with microtubules.

Age of Onset↗
PubMed1998

Characterization of the mouse Prox1 gene.

Prox1, a vertebrate homologue of Drosophila prospero, encodes a divergent homeodomain protein. We have isolated and characterized full length mouse Prox1 cDNA and genomic clones. Mouse Prox1 gene mapped to position 106.3 cM from the centromere of Chromosome 1, which is very close to the retinal degeneration mutation, rd3. Although the coding sequence and exon-intron junctions of the Prox1 genes of wild type and rd3 mutant mice are identical, Northern blot analysis indicated that the ratio of the short (2.3 kb) and long (8 kb) forms of Prox1 mRNA is different in RNA isolated from wild type and rd3 retinas. Immunostaining of the eyes from wild type and rd3 animals also revealed differences in the distribution of Prox1 protein in the retina and lens. These data suggest that the rd3 mutation affects expression of the mouse Prox1 gene.

Amino Acid Sequence↗
PubMed1998

Characterization of the mouse Myoc/Tigr gene.

Mutations in the myocilin (MYOC), also known as Trabecular meshwork-Inducible Glucocorticoid Response (TIGR) gene can lead to juvenile open-angle glaucoma in human and may be responsible for at least 3% of primary open-angle glaucoma. To develop a mouse model of primary open angle glaucoma, and to get deeper insight into the mechanisms of the MYOC/TIGR gene regulation and function, we have isolated and characterized full size mouse Myoc/Tigr cDNA and genomic clones. The mouse and human MYOC/TIGR genes have the same exon-intron structure and contain 3 exons, although the mouse gene is 6 kb shorter than the human gene (10 kb versus 16 kb) due to differences in the length of introns. The MYOC/TIGR gene encodes a moderately conserved protein, which is 82% identical between human and mouse. The encoded protein is 14 amino acids shorter at the N-terminus in the mouse than in the human (490 versus 504 amino acids). Mouse and human MYOC/TIGR genes show a similar pattern of expression in adult ocular and nonocular tissues. The mouse Myoc/Tigr gene was mapped to Chromosome 1 at position 82.8 cM from the centromere. All residues, which were identified in the human MYOC/TIGR protein as critical for glaucoma development, are conserved in the mouse Myoc/Tigr.

Adult↗
PubMed1998

Restricted expression of the homeobox gene prox 1 in developing zebrafish.

Prox 1 is a vertebrate homeobox gene which is homologous to the Drosophila transcription factor, prospero. We have isolated a prox 1 cDNA from zebrafish, which encodes a protein that has 82%, 84% and 83% amino acid identity with chicken, mouse and human Prox 1, respectively. Antibodies raised against human Prox 1 cross-react with zebrafish Prox 1 and are used here to determine the expression patterns of Prox 1 during zebrafish embryogenesis by whole-mount immunohistochemistry. In the 10-somite embryo, Prox 1 is expressed over the prospective lens placode and over a broad region of epithelium extending from the eye to the otic vesicle. As embryogenesis proceeds, Prox 1 expression in the eye lens becomes intense, and is detected in maturing muscle pioneer cells and superficial muscle cells. In the CNS, Prox 1 is expressed in a stripe along the forebrain-midbrain boundary, in a segmented pattern in the ventral hindbrain, and in selected cells of the ventral spinal cord. Additional sites of Prox 1 expression include the lateral line primordium, the trigeminal ganglia, the otic vesicle and occasional endodermal cells.

Amino Acid Sequence↗
PubMed1997

Squid Pax-6 and eye development.

Pax-6 in vertebrates and its homolog eyeless in Drosophila are known to be essential for eye development. Here we investigate the role of Pax-6 in eye development in another major systematic group, molluscs. We demonstrate that alternatively spliced RNAs derived from a single Pax-6 gene in the squid (Loligo opalescens) are expressed in the embryonic eye, olfactory organ, brain, and arms. Despite significant sequence differences between squid Pax-6 and Drosophila eyeless in the region outside the paired- and homeodomains, squid Pax-6 is able to induce the formation of ectopic eyes in Drosophila. Our results support the idea that Pax-6 related genes are necessary for eye and olfactory system formation throughout the animal kingdom.

Amino Acid Sequence↗
PubMed1997

Eyes absent: a gene family found in several metazoan phyla.

Genes related to the Drosophila eyes absent gene were identified in vertebrates (mouse and human), mollusks (squid), and nematodes (C. elegans). Proteins encoded by these genes consist of conserved C-terminal and variable N-terminal domains. In the conserved 271-amino acid C-terminal region, Drosophila and vertebrate proteins are 65-67% identical. A vertebrate homolog of eyes absent, designated Eya2, was mapped to Chromosome (Chr) 2 in the mouse and to Chr 20q13.1 in human. Eya2 shows a dynamic pattern of expression during development. In the mouse, expression of Eya2 was first detected in 8.5-day embryos in the region of head ectoderm fated to become the forebrain. At later stages of development, Eya2 is expressed in the olfactory placode and in a variety of neural crest derivatives. In the eye, expression of Eya2 was first detected after formation of the lens vesicle. At day 17.5, the highest level of Eya2 mRNA was observed in primary lens fibers. Low levels of Eya2 expression was detected in retina, sclera, and cornea. By postnatal day 10, Eya2 was expressed in secondary lens fibers, cornea, and retina. Although Eya2 is expressed relatively late in eye development, it belongs to the growing list of factors that may be essential for eye development across metazoan phyla. Like members of the Pax-6 gene family, eyes absent gene family members were probably first involved in functions not related to vision, with recruitment for visual system formation and function occurring later.

Age Factors↗