The role of Rho GTPases in the regulation of the rearrangement of actin cytoskeleton and cell movement.

The rearrangement of the actin cytoskeleton has been shown to play a critical role in the development of transformation and malignant phenotype of cancer cells. Rho family GTPases regulate the arrangement of the actin cytoskeleton. By wound-healing assay, we have found that NIH 3T3 fibroblast cells move towards the wound- gaps by extending filopodial and lamellipdial structures at the leading edge of the moving cells.

Identification of a neurite outgrowth-promoting motif within the alternatively spliced region of human tenascin-C.

Our work centers on understanding how the extracellular matrix molecule tenascin-C regulates neuronal growth. We have found that the region of tenascin-C containing only alternately spliced fibronectin type-III repeat D, called fnD, when used by itself, dramatically increases neurite outgrowth in culture. We used overlapping synthetic peptides to localize the neurite outgrowth-promoting site within fnD to a 15 amino acid sequence, called D5.

Localisation of a gene for an autosomal recessive syndrome of macrocephaly, multiple epiphyseal dysplasia, and distinctive facies to chromosome 15q26.

Background: We have previously described an autosomal recessive syndrome of macrocephaly, multiple epiphyseal dysplasia (MED), and distinctive facies in a large, extended Omani family. The MED observed seems to be part of a larger malformation syndrome, since both craniofacial and central nervous system changes were present in the family. We performed a whole genome scan in this family in order to identify the gene locus for this malformation syndrome.

Differential requirement for Rho family GTPases in an oncogenic insulin-like growth factor-I receptor-induced cell transformation.

Insulin-like growth factor I receptor (IGFR) plays an important role in cell growth and transformation. We dissected the downstream signaling pathways of an oncogenic variant of IGFR, Gag-IGFR, called NM1. Loss of function mutants of NM1, Phe-1136 and dS2, that retain kinase activity but are attenuated in their transforming ability were used to identify signaling pathways that are important for transformation of NIH 3T3 cells.

Serum-dependent perinuclear accumulation of Cdc42 in mammalian cells.

Cdc42 is a member of the Rho family of GTPase. Cdc42 has been implicated to be involved in the movement, multiplication and transformation of mammalian cells by controlling the rearrangement of actin cytoskeleton and gene expression. But the mechanism of Cdc42 function has not yet been discovered.

Proto-oncogene ras GTPase-linked induction of glutathione-S-transferase by growth factors in PC12 cells.

This report provides evidence linking activation of Ras GTPase by growth factors and induction of glutathione-S-transferase isozymes in PC12 cells. Ras GTPase was activated by EGF, NGF, insulin and phorbolester in PC12 cells. Activation of Ras GTPase was found to be associated with induction of the expression of GST mu and pi isoenzymes while there was no detectable induction of GST alpha expression.

Cytoskeletal tumor suppressors that block oncogenic RAS signaling.

Several distinct peptides or drugs that block the Rho family GTPases-mediated pathways were found to suppress RAS-induced malignant phenotype. They include (1) C3 enzyme that selectively inactivates Rho, (2) ACK42, a peptide that blocks the interaction of CDC42 with its effectors such as ACKs, (3) PAK18, a peptide that blocks the activation of PAK and membrane ruffling, and (4) actin-binding drugs, chaetoglobosin K (CK) and MKT-077, that block membrane ruffling by capping and bundling actin filaments, respectively.

The CDC42-specific inhibitor derived from ACK-1 blocks v-Ha-Ras-induced transformation.

Based on the previous experiments with the N17 mutant of CDC42, it has been speculated, but not proved as yet, that CDC42 is required for Ras-induced malignant transformation of fibroblasts. However, since this inhibitor could sequester many GDP-dissociation stimulators (GDSs), such as DBL, OST and Tiam-1 which activate not only CDC42, but also Rho or Rac, in fact it is not a specific inhibitor that inactivates only CDC42. Thus, we have taken the minimum CDC42-binding domain (residues 504 - 545, called ACK42) of the Tyr-kinase ACK-1 that binds only CDC42 in the GTP-bound form, and thereby blocking the interactions of CDC42-GTP with its downstream effectors such as ACKs, PAKs and N-WASP.

Homozygosity mapping in families with Joubert syndrome identifies a locus on chromosome 9q34.3 and evidence for genetic heterogeneity.

Joubert syndrome is a rare developmental defect of the cerebellar vermis, with autosomal recessive inheritance. The phenotype is highly variable and may include episodic hyperpnea, abnormal eye movements, hypotonia, ataxia, developmental delay, and mental retardation. Even within sibships the phenotype may vary, making it difficult to establish the exact clinical diagnostic boundaries of Joubert syndrome.

G proteins, phosphoinositides, and actin-cytoskeleton in the control of cancer growth.

Almost three decades have passed since actin-cytoskeleton (acto-myosin complex) was first discovered in non-muscle cells. A combination of cell biology, biochemistry, and molecular biology has revealed the structure and function of many actin-binding proteins and their physiological role in the regulation of cell motility, shape, growth, and malignant transformation. As molecular oncologists, we would like to review how the function of actin-cytoskeleton is regulated through Ras/Rho family GTPases- or phosphoinosites-mediated signaling pathways, and how malignant transformation is controlled by actin/phosphoinositides-binding proteins or drugs that block Rho/Rac/CDC42 GTPases-mediated signaling pathways.

Tenascin-C contains domains that independently regulate neurite outgrowth and neurite guidance.

Tenascin-C has been implicated in regulation of both neurite outgrowth and neurite guidance. We have shown previously that a particular region of tenascin-C has powerful neurite outgrowth-promoting actions in vitro. This region consists of the alternatively spliced fibronectin type-III (FN-III) repeats A-D and is abbreviated fnA-D.

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4.

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol.

Comparative studies on the sensitivity of polymerase chain reaction and microscopic examination for the detection of Trypanosoma evansi in experimentally infected mice.

Trypanosoma evansi, a protozoan parasite in the blood of camels is routinely diagnosed by finding the flagellates in the wet films or stained smear of peripheral blood, examined under a microscope. Although specific, this method is not sensitive at early stages of infection. We have tested the use of polymerase chain reaction (PCR) in the identification of T.

United Arab Emirates population data on three DNA tetrameric short tandem repeat loci: HUMTHO1, TPOX and CSF1PO--derived using multiplex polymerase chain reaction and manual typing.

Allele and genotype frequencies for three tetrameric short tandem repeat (STR) loci: HUMTHO1, TPOX, and CSFIPO, were determined in a United Arab Emirates (UAE) national population sample (n = 119). The loci were amplified simultaneously and the PCR products were separated in polyacrylamide DNA sequencing gels. Detection of the DNA fragments was accomplished using silver staining.

The GTPase and Rho GAP domains of p190, a tumor suppressor protein that binds the M(r) 120,000 Ras GAP, independently function as anti-Ras tumor suppressors.

p190 is a Tyr-phosphorylatable G protein of M(r) 190,000 that binds NH2-terminal SH2 domains of GAP1, a Ras GAP of M(r) 120,000. p190 contains at least two functional domains: a GTPase domain at the NH2 terminus and a GAP domain at the COOH terminus that can attenuate signal-transducing activity of three distinct G proteins (Rac, Rho, and CDC42). Here, we demonstrate that overexpression of either an antisense p190 RNA or a dominant negative mutant (Asn36) of p190 GTPase domain (residues 1-251) but not the wild-type p190 GTPase domain is able to transform normal NIH/3T3 fibroblasts.

Targeted delivery of human neurofibromin and c-Raf-1 mutants to the cytoplasmic membrane by use of the influenza virus hemagglutinin.

Mutants of human neurofibromin and c-Raf-1 genes were fused to the 3' end of the hemagglutinin (HA) gene of influenza A virus by oligonucleotide-directed polymerase chain reaction (PCR). The two resulting chimeric genes, HA (1-534)/NF1 (1441-1518) and HA (1-534)/Raf-1 (51-132) which we designated HN and HR, respectively, were cloned in a vaccinia virus expression vector (pTMI) under the control of a T7 RNA polymerase promoter. The clones were expressed in a monkey cell line (CV-1) and the resulting chimeric proteins analysed.

Overexpression and functional analysis of a mitogen-inducible nuclear GTPase activating protein, Spa-1.

The two Ras-related GTPases called Rap1 and Rsr1, which share 50% sequence identity with Ras GTPases are known to be activated by two distinct mammalian GAPs, i.e. cytosolic GAP3c of 55 kDa and membrane-bound GAP3m of 85 kDa.

Molecular characterization of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Al-Ain District, United Arab Emirates.

In a cross-sectional study, the activity, electrophoretic mobility and genotypes of glucose-6-phosphate dehydrogenase (G6PD) were determined among healthy, UAE national school boys from Al-Ain District in the United Arab Emirates, The prevalence of G6PD deficiency in this population sample was 11%. The majority of G6PD-deficient subjects were descendants of Omani, Baluchi or Yemeni migrants. Of 18 deficient subjects, 16 had an enzyme activity of < 10% of normal while 2 had an activity of just above 10%.

Characterization of a synthetic peptide mimicking trypsin-cleavage site of rotavirus VP4.

A synthetic peptide corresponding to the trypsin cleavage site on the 84 k protein of bovine rotavirus was synthesized (VP4-peptide). This synthetic peptide could be cleaved by trypsin and therefore possessed the enzyme binding site present on the authentic protein. Further proof that this peptide mimicks the authentic trypsin cleavage site was the specific reaction of anti-peptide serum with the 84 k protein.

Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell cycle progression.

We have cloned a novel cDNA (Spa-1) which is little expressed in the quiescent state but induced in the interleukin 2-stimulated cycling state of an interleukin 2-responsive murine lymphoid cell line by differential hybridization. Spa-1 mRNA (3.5 kb) was induced in normal lymphocytes following various types of mitogenic stimulation.

The minimal fragments of c-Raf-1 and NF1 that can suppress v-Ha-Ras-induced malignant phenotype.

v-Ha-Ras, an oncogenic Ras mutant, causes malignant transformation of mammalian cells by recruiting c-Raf-1, a cytosolic Ser/Thr kinase, to the plasma membranes/cytoskeleton. The kinase activity of c-Raf-1 resides in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf257, residues 1-257) that binds the Ras-GTP complex and can compete Ras GTPase-activating proteins such as NF1 for binding to Ras. However, it still remains to be clarified whether overexpression of Raf257 or its minimal Ras-binding fragment alone is sufficient to suppress Ras-induced malignancy.

Molecular cloning of the wild-type and mutant thyA gene from Shigella flexneri Y.

The thyA gene which codes for thymidylate synthase has been cloned and sequenced from the wild-type Shigella flexneri Y strain SH4 and a thyA mutant TSF21 after amplifying the gene by polymerase chain reaction (PCR). The nucleotide sequence revealed 98% homology to the E. coli K-12 thyA gene.

rho GAP of 28 kDa (GAP2), but not of 190 kDa (p190), requires Asp65 and Asp67 of rho GTPase for its activation.

Two distinct GTPase-activating proteins (GAPs), i.e. rho GAPs of 28 kDa (GAP2) and of 190 kDa (p190), stimulate the intrinsic GTPase activity of the rho protein.

The GTPase-activating NF1 fragment of 91 amino acids reverses v-Ha-Ras-induced malignant phenotype.

The human neurofibromatosis type 1 gene encodes a Ras GAP (GTPase-activating protein) of 2818 amino acids called NF1. This GAP contains a domain of 338 amino acids (residues 1194-1531) called NF1-GRD, which shares 26% sequence identity with the C-terminal domain (GAP1C, residues 709-1044) of another Ras GAP called GAP1. Both NF1-GRD and GAP1C activate normal Ras GTPases but not oncogenic mutants such as v-Ha-Ras.