The EuroPhysiome, STEP and a roadmap for the virtual physiological human.

Biomedical science and its allied disciplines are entering a new era in which computational methods and technologies are poised to play a prevalent role in supporting collaborative investigation of the human body. Within Europe, this has its focus in the virtual physiological human (VPH), which is an evolving entity that has emerged from the EuroPhysiome initiative and the strategy for the EuroPhysiome (STEP) consortium. The VPH is intended to be a solution to common infrastructure needs for physiome projects across the globe, providing a unifying architecture that facilitates integration and prediction, ultimately creating a framework capable of describing Homo sapiens in silico. The routine reliance of the biomedical industry, biomedical research and clinical practice on information technology (IT) highlights the importance of a tailor-made and robust IT infrastructure, but numerous challenges need to be addressed if the VPH is to become a mature technological reality. Appropriate investment will reap considerable rewards, since it is anticipated that the VPH will influence all sectors of society, with implications predominantly for improved healthcare, improved competitiveness in industry and greater understanding of (patho)physiological processes. This paper considers issues pertinent to the development of the VPH, highlighted by the work of the STEP consortium.

Similarities and differences in the subcellular localization of hamartin and tuberin in the kidney.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in multiple organs, notably the brain and kidneys. The disease is caused by mutations in TSC1 or TSC2 genes, coding hamartin and tuberin, respectively. Immunofluorescence analysis of tuberin and hamartin performed here demonstrates that both proteins are specifically expressed in the distal urinary tubule, comprising the distal tubules, connecting segment, and collecting ducts. Hamartin, distinct from tuberin, is expressed in the thick ascending limbs of Henle and in juxtaglomerular cells, where it colocalizes with renin. In positive epithelial cells, tuberin localizes to the cytoplasm as well as the apical membrane. Hamartin, however, preferentially localizes to the apical membrane. The two proteins colocalize at the apical membrane of type A intercalated cells and connecting tubule cells, whereas in type B intercalated cells they reveal a variable pattern of expression. The cell-specific expression of tuberin and hamartin described here will provide critical insight into the cell types that give rise to kidney lesions, and the tumor suppressor role of these proteins in TSC.

Analysis of molecular domains of epitope-tagged merlin isoforms in Cos-7 cells and primary rat Schwann cells.

The Neurofibromatosis 2 gene product, merlin, has striking similarity to ezrin, radixin, and moesin (ERM), members of the protein 4.1 family which have been demonstrated to connect proteins in the plasma membrane to the cytoskeletal components. The recent localization of merlin to the motile regions in cultured cells such as membrane ruffles further supports the notion that merlin represents a new class of tumor suppressors. Here we describe the localization of full-length and truncated polypeptides of merlin expressed as Flag-tagged proteins in transfected cells. Similar to endogenous merlin, the epitope-tagged full-length merlin localizes to the membrane ruffles in transfected Cos-7 cells and rat Schwann cells. In addition, the over-expressed merlin localizes to other actin-rich cortical structures, such as microvilli and filopodia. The amino-terminal half of merlin is seen dispersed throughout the cells and in membrane ruffles. Compared to the amino-terminal half of merlin, its carboxy-terminal half localizes more distinctly to membrane ruffles. The full-length and the carboxy-terminal portion of merlin co-localize with F-actin at the membrane ruffles. However, distinct from the ERM proteins, the carboxy-terminal-truncated merlin and F-actin do not co-localize with each other at the stress fibers. Our results suggest that both the amino- and the carboxy-terminal domains of merlin contribute to its membrane ruffle localization.

NHE-RF, a regulatory cofactor for Na(+)-H+ exchange, is a common interactor for merlin and ERM (MERM) proteins.

We have identified the human homologue of a regulatory cofactor of Na(+)-H+ exchanger (NHE-RF) as a novel interactor for merlin, the neurofibromatosis 2 tumor suppressor protein. NHE-RF mediates protein kinase A regulation of Na(+)-H+ exchanger NHE3 to which it is thought to bind via one of its two PDZ domains. The carboxyl-terminal region of NHE-RF, downstream of the PDZ domains, interacts with the amino-terminal protein 4.1 domain-containing segment of merlin in yeast two-hybrid assays. This interaction also occurs in affinity binding assays with full-length NHE-RF expressed in COS-7 cells. NHE-RF binds to the related ERM proteins, moesin and radixin. We have localized human NHE-RF to actin-rich structures such as membrane ruffles, microvilli, and filopodia in HeLa and COS-7 cells, where it co-localizes with merlin and moesin. These findings suggest that hNHE-RF and its binding partners may participate in a larger complex (one component of which might be a Na(+)-H+ exchanger) that could be crucial for the actin filament assembly activated by the ERM proteins and for the tumor suppressor function of merlin.

Universal absence of merlin, but not other ERM family members, in schwannomas.

NF2 (neurofibromatosis 2, encoding the merlin protein) gene mutations and chromosome 22q loss have been demonstrated in the majority of sporadic and NF2-associated schwannomas, but many schwannomas fail to demonstrate genetic evidence of biallelic NF2 gene inactivation. In addition, the role of the merlin-related ERM family members (ezrin, radixin, and moesin) remains unclear in these tumors. We therefore studied expression of NF2-encoded merlin as well as ezrin, radixin, and moesin in 22 vestibular and peripheral schwannomas that had been evaluated for NF2 mutations and chromosome 22q loss. Western blotting and immunohistochemistry with antibodies directed against the amino and carboxy termini of merlin demonstrated loss of merlin expression in all studied schwannomas, including 12 tumors lacking genetic evidence of biallelic NF2 gene inactivation. Western blotting with antibodies directed against ezrin, radixin, and moesin, however, showed expression of these proteins in all schwannomas. In addition, immunohistochemistry with an antibody to moesin revealed widespread expression in tumor and endothelial cells. These data indicate that the specific loss of merlin is universal to schwannomas and is not linked to loss of ezrin, radixin, or moesin expression.

Expression of NF2-encoded merlin and related ERM family proteins in the human central nervous system.

Germline mutations of the neurofibromatosis 2 (NF2) gene are associated with an increased incidence of gliomas and glial harmartomas, suggesting a role for the NF2-encoded protein, merlin, in glial growth control. Using monoclonal and polyclonal anti-merlin antibodies for Western blotting and immunohistochemistry, we evaluated the cellular pattern of merlin expression in the normal human central nervous system (CNS), reactive gliosis; and NF2-associated glial hamartomas. In the normal CNS, merlin is widely expressed in coarse cytoplasmic granules in both glia and neurons, with less pronounced expression in other cells. Merlin is also expressed in reactive astrocytes and in the astrocytes of NF2-associated glial hamartomas. In reactive astrocytes, however, merlin is also present at the cell membrane and in cellular processes, suggesting redistribution of the protein in activated cells. Merlin is structurally related to ezrin, radixin and moesin, which are also expressed in the CNS, as demonstrated by Western blotting. The pattern of merlin expression, however, is distinct from that of ezrin, which has been previously described, and that of moesin, in which immunohistochemistry with an anti-moesin antibody showed expression in endothelial cells, glia and neurons in a membranous or diffuse cytoplasmic pattern. These findings imply that merlin has widespread and specific functions in the human central nervous system.

The merlin tumor suppressor localizes preferentially in membrane ruffles.

Merlin is a tumor suppressor whose inactivation underlies the familial schwannomas and meningiomas of neurofibromatosis 2 and their sporadic counterparts. It bears striking similarity to the ERM proteins, ezrin, radixin and moesin, members of the protein 4.1 superfamily that link proteins in the cytoskeleton and the plasma membrane. We have generated polyclonal and monoclonal antibodies that detect merlin as an approximately 66 kD protein in many different cell types. Using indirect immunofluorescence we have for the first time visualized endogenous merlin and localized it to the motile regions, such as leading or ruffling edges, in human fibroblast and meningioma cells. Merlin co-localizes with F-actin in these motile regions but is not associated with stress fibers. Merlin does not localize to the same structures as either ezrin or moesin in human meningioma cells, suggesting a function distinct from these ERMs. Thus, merlin is associated with motile regions of the cell and its participation in these structures may be intimately involved in control of proliferation in Schwann cells and meningeal cells.

Neurofibromatosis 2 gene in human colorectal cancer.

Colon cancers commonly have allelic losses of chromosome 22q, which suggests the presence of a tumor suppressor gene on 22q. The candidate tumor suppressor gene on 22q is the neurofibromatosis 2 (NF2) gene. Using single strand conformation polymorphism (SSCP) analysis, we screened 24 pairs of colorectal cancer and adjacent normal mucosa, as well as 10 colon cancer cell lines from non-NF2 patients, for mutations in the coding sequence of the NF2 gene. Two SSCP variants, one in exon 14 and another one in exon 16, were detected in two of the sporadic colorectal cancers, but not in adjacent normal mucosa samples. Sequencing of these variants in one tumor detected an A-to-G transition in bp 1459 of the NF2 cDNA, resulting in the change of Ile to Val at codon 487 of merlin, the NF2 protein product. The other tumor showed a 2-bp (CT) deletion in the intronic sequence of the alternatively spliced exon 16. These results suggest that the NF2 gene is probably involved in some colorectal tumors, but is not the critical chromosome 22q tumor suppressor gene involved in colon tumorigenesis.

Quail myoD is regulated by a complex array of cis-acting control sequences.

Quail myoD (QmyoD) is the earliest myoD family member expressed in quail somites and its transcription is initiated in response to early developmental signals. We have investigated the transcriptional regulation of QmyoD to define the cis-acting sequences required for tissue-specific and correct developmental expression. The QmyoD gene locus was isolated and sequenced and its regulatory properties were characterized. We identified three distinct regions of cis-acting regulatory sequences that control the expression of reporter gene constructs following DNA transfection into cell lines and cultured primary quail cells. The first, a complex distal control region (DCR), 11.5 kb upstream of the gene, contains three separable enhancer activities. Two of these DCR enhancer activities are tissue specific and can be autoactivated. In addition, these same two enhancers and the entire DCR direct somite- and muscle-specific expression of a reporter gene in transgenic mice. Sequence analysis of the DCR enhancers reveals clusters of E-boxes, MEF2 binding motifs, and the stretches of sequence identity with the human myoD enhancer. Second, the promoter region has sequences which act positively to direct expression in both muscle and nonmuscle cells as well as sequences that repress expression specifically in nonmuscle cells. The third control region, the PR, is located -3.3 to -5 kb from the transcription start site and directs muscle-specific expression in cultured cells. This analysis demonstrates that QmyoD has multiple control regions and that some features of myoD regulation are conserved between mammals and birds.

Microvascular permeability and endothelial cell morphology associated with low-flow ischemia/reperfusion injury in the equine jejunum.

Microvascular permeability of the jejunum of clinically normal equids and microvascular permeability associated with 60 minutes of ischemia (25% baseline blood flow) and subsequent reperfusion were investigated. Eight adult horses were randomly allotted to 2 equal groups: normal and ischemic/reperfusion injury. Lymphatic flow rates, mesenteric blood flow, and lymph and plasma protein concentrations were determined at 15-minute intervals throughout the study. Microvascular permeability was determined by estimates of the osmotic reflection coefficient, which was determined when the ratio of lymphatic protein to plasma protein concentration reached a constant minimal value as lymph flow rate increased (filtration-independent lymph flow rate), which occurred at venous pressure of 30 mm of Hg. Full-thickness jejunal biopsy specimens were obtained at the beginning and end of each experiment, and were prepared for light microscopy to estimate tissue volume (edema) and for transmission electron microscopy to evaluate capillary endothelial cell morphology. The osmotic reflection coefficient for normal equine jejunum was 0.19 +/- 0.06, and increased significantly (P < or = 0.0001) to 0.48 +/- 0.05 after the ischemia/reperfusion period. Microscopic evaluation revealed a significant increase (P < or = 0.0001) in submucosal and serosal volume and capillary endothelial cell damage in horses that underwent ischemia/reperfusion injury. Results indicate that ischemia/reperfusion of the equine jejunum caused a significant increase in microvascular permeability.

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas.

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.

Continuous versus intermittent epidural analgesia. A randomised trial to observe obstetric outcome.

A randomised study of 381 women was carried out to compare the obstetric outcome after epidural analgesia maintained by an intermittent top-up regimen or with a continuous infusion. The two groups were well matched with respect to age, parity, mode of onset of labour and indication for epidural. Maintenance of epidural analgesia by continuous infusion resulted in a significantly decreased need for top-up doses. A reduction in the incidence of hypotension, cardiotocographic evidence of intrapartum fetal hypoxia and Caesarean section was associated with this. It is concluded that the maintenance of epidural analgesia by continuous infusion is a safe and reliable method and may be more advantageous and less labour intensive than the traditional intermittent regimen.

10T1/2 cells: an in vitro model for molecular genetic analysis of mesodermal determination and differentiation.

Progress has been made in understanding the molecular mechanisms that regulate cell type-specific gene expression during the terminal differentiation of cells into specialized tissue types. These studies have concentrated largely on defining the cis elements and trans-acting factors responsible for the transcription of differentiation-specific genes. Valuable as these investigations have been, they have not been able to place differentiation into the larger context of development, specifically into the context of the earlier developmental process of cell determination, when embryonic stem cell lineages are formed and the genetic regulatory programs for cell type-specific gene activation and expression are acquired by stem cells. The clonal mouse embryo cell line, C3H/10T1/2, clone 8 (10T1/2) provides a unique opportunity to examine the molecular genetic regulation of both the developmental determination of vertebrate stem cell lineages and their subsequent differentiation. 10T1/2 is an apparently multipotential cell line that can be converted by 5-azacytidine into three mesodermal stem cell lineages. These determined proliferative stem cells are stable in culture and retain their ability to differentiate in mitogen-depleted medium. The most significant discovery has been that 10T1/2 lineage determination is under simple genetic control and that the regulatory genes that mediate the formation of myogenic cell lineages, and likely the chondrogenic and adipogenic lineages, can be demonstrated and studied by genomic DNA and cDNA transfection approaches. This paper is a description of the remarkable properties and genetic behaviors of the 10T1/2 cells and a discussion of the insights that future studies of this cell may provide.

Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway.

Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene. The myogenic phenotype remains linked to neomycin resistance during secondary transfections. Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins. We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage. Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene. Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not. These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation.

Identification and characterization of a group of discrete initiated oligonucleotides transcribed in vitro from the 3' terminus of the N-gene of vesicular stomatitis virus.

Four triphosphate-initiated oligonucleotides, 11 to 14 bases long, produced by in vitro transcription of vesicular stomatitis virus were identified as the uncapped 5' sequences of N-gene mRNA. Characterization of these oligonucleotides reveals that they are continually produced stable transcripts that do not remain template bound. Under the conditions used, the oligonucleotide transcripts were produced at 8 to 10 times the molar amount of leader, suggesting that the N-gene mRNA is internally initiated.

In vitro synthesis of triphosphate-initiated N-gene mRNA oligonucleotides is regulated by the matrix protein of vesicular stomatitis virus.

Wild-type Indiana virus transcribed four 11- to 14-nucleotide-long, 5' N-gene mRNA sequences in vitro. The amount of oligonucleotides synthesized relative to leader by wild-type virions varied inversely with the salt concentration of the transcription reaction. Reduced oligonucleotide synthesis by nucleocapsids at all salt concentrations tested and a comparison of the proteins remaining bound to the template of nucleocapsids and virions transcribed in different NaCl concentrations suggested that the matrix (M) protein regulates oligonucleotide synthesis. Examination of the transcription products synthesized in no NaCl and 0.144 M NaCl by an M-protein mutant and an increase in oligonucleotide synthesis by nucleocapsids when purified M-protein was added to transcription reactions confirmed M-protein's role in oligonucleotide synthesis. Wild-type virion mRNA synthesis was inhibited, and oligonucleotide synthesis was greater than leader synthesis at high virus concentrations. As the virus was diluted, inhibition of mRNA synthesis was relieved and oligonucleotide synthesis was reduced. The M-protein mutant tsG33 exhibited neither transcription inhibition at high virus concentrations nor the reciprocal synthesis of mRNA and the oligonucleotides seen with wild-type virions. These results are entirely consistent with the stop-start model of transcription and suggest a model for the control of transcription by M-protein.

Changing erythrocyte populations in juvenile chronic myelocytic leukemia: evidence for disordered regulation.

Fetal and adult erythrocyte characteristics were studied serially in a 30-mo-old female with juvenile chronic myelocytic leukemia. On presentation the erythrocytes exhibited predominantly fetal characteristics as indicated by 69% hemoglobin F (HbF), 1.1% hemoglobin A2 (HbA2), absent I antigen, and fetal levels of the erythrocyte enzymes, carbonic anhydrase I and II, glucose-6-phosphate dehydrogenase, hexokinase, pyruvate kinase, and lactate dehydrogenase; 100% of the erythrocytes present contained HbF. However, Orskov-Jacobs-Stewart hemolysis demonstrated that at least one adult characteristic was present. Seven months later HbF was 17%; I antigen and carbonic anhydrase I had increased to adult levels. The number of cells containing HbF had decreased to 30%. Further studies indicated that at least three new populations of red cells were present after 7 mo which had not previously been detected. Two of these populations exhibited a mixture of both fetal and adult characteristics. Such findings suggested that an ongoing disturbance of regulatory mechanisms was responsible for the variable expression of fetal versus adult erythrocyte characteristics.