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Biomedical subjects

N Usman

Publications and source records attributed to N Usman.

At least 19 recordsLinked to original sources
PubMed2006

RhoA and RhoC proteins promote both cell proliferation and cell invasion of human oesophageal squamous cell carcinoma cell lines in vitro and in vivo.

There is growing evidence that Rho proteins are deregulated by overexpression in tumours; and according to some reports, this correlates with disease progression. Our previous clinical study had demonstrated a correlation between RhoA expression and tumour progression in oesophageal squamous cell carcinoma (ESCC). These findings prompted us to study, using nude mice, pathological roles of Rho proteins in human ESCC cells. Western blot analysis in ESCC cell lines, in addition to cell proliferation and in vitro migration assays, were performed to observe the malignant potential of RhoA and RhoC in untransfected and transfected cells. Constitutively active RhoA, RhoC and dominant negative RhoA (dnRhoA) proteins were transfected to ESCC (TE-1 and TE-2) cells. The stably transfected cells were injected into nude mice, and the growth and metastasis of these cells to the lungs were analysed. Tumour tissues were then examined using immunohistochemical methods for proteins Ki-67 (MIB-1), FAK, MMP-1, MMP-9 and TIMP-3. Protein levels of RhoA and RhoC in ESCC cell lines were visualised by Western blotting, and showed highest expression in TE-2 cells. Results from the migration assay illustrated that both RhoA and RhoC play a role in migration of ESCC cells. In TE-2 transfected cells, RhoC showed greater migration compared to RhoA. By using an experimental metastasis model in nude mice, RhoA was found to promote more tumour growth than RhoC, whereas RhoC induced lung metastasis in comparison to RhoA. Ki-67 labelling index was used to evaluate the proliferation potential of tumour tissue inoculated from nude mice. In TE-2 cells RhoA gave a proliferation capacity of 24.8+/-0.5, which was significantly higher than those of TE-2 RhoC 10+/-0.4 (P<0.01). Strong immunoreactivity for FAK, MMP-1 and MMP-9 proteins was present in all tumour cells. By contrast, loss of TIMP-3 expression was observed in all tumour cells. In conclusion, our results indicate that pro-oncogenic Rho proteins are involved in promoting tumour growth, cell migration and metastasis in human ESCC cells in nude mice. The results from this study suggest that active Rho proteins may induce a transforming effect that leads to a malignant phenotype.

Animals↗
PubMed2002

Perceptions of influenza and influenza vaccination in patients attending hospital outpatient clinics.

Influenza remains a major cause of morbidity and disruption to health services during winter. Annual vaccination of risk groups is the mainstay of influenza management policy. Despite much national publicity, vaccine uptake remains below desired levels. We investigated the perceptions of influenzal illness and opinions on vaccination in hospital outpatients attending clinics associated with risk and non-risk (i.e. control) morbidities. There were 270 recruited patients in risk groups: 90 each from diabetic, respiratory and cardiac clinics and equal numbers in three age groups (40-49, 50-59, 60-69 years). Equal numbers of controls were recruited from colorectal, fracture and dermatology clinics. The perceptions of influenza and knowledge of the preventive value of vaccination were fairly accurate in both groups. Fewer people in both groups had been vaccinated than had been specifically advised to be vaccinated. Advice for vaccination had been received by fewer people attending the cardiac clinic than the other two risk associated clinics. More use could be made of the health education opportunity afforded by attendance at hospital outpatient clinics.

Adult↗
PubMed2001

Angiozyme: a novel angiogenesis inhibitor.

Several inhibitors of angiogenesis are being developed for the treatment of cancer. One dominant strategy involves disruption of the vascular endothelial growth factor (VEGF) pathway by inhibition of the receptors for VEGF. Inhibition of the VEGF receptor activity can be accomplished using catalytic RNA molecules known as ribozymes, which downregulate VEGF receptor function by specifically cleaving the mRNAs for the primary VEGF receptors, Flt-1 and KDR. Significant inhibition of angiogenesis using ribozymes against both receptors has been demonstrated. In animal tumor models, antitumor effects are most pronounced with the anti-Flt-1 ribozyme known as Angiozyme (Ribozyme Pharmaceuticals, Boulder, CO). Extensive preclinical studies have demonstrated no significant toxicities. Clinical trials of Angiozyme are currently in progress for patients with advanced malignancy. Preliminary results demonstrate Angiozyme to be well tolerated, without significant side effects. Several phase II trials are underway for patients with advanced malignancy to test therapeutic efficacy.

Angiogenesis Inhibitors↗
PubMed2001

X-ray crystallographic observation of "in-line" and "adjacent" conformations in a bulged self-cleaving RNA/DNA hybrid.

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Hüsken et al., Biochemistry, 1996, 35:16591-16600). Transesterification proceeds via an in-line mechanism, with the 2'-OH of the bulged nucleotide attacking the 3'-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2'-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2'-oxygen of the bulged nucleotide and the P-O5' bond (including adjacent and near in-line) and give a detailed picture of the conformational changes necessary to line up the 2'-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308-1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.

Adenosine↗
PubMed2001

Cortical upper layer neurons derive from the subventricular zone as indicated by Svet1 gene expression.

The cerebral cortex is composed of a large variety of different neuron types. All cortical neurons, except some interneurons, are born in two proliferative zones, the cortical ventricular (VZ) and subventricular (SVZ) zones. The relative contribution of both proliferative zones to the generation of the diversity of the cortical neurons is not well understood. To further dissect the underlying mechanism, molecular markers specific for the SVZ are required. Towards this end we performed a subtraction of cDNA libraries, generated from E15.5 and E18.5 mouse cerebral cortex. A novel cDNA, Svet1, was cloned which was specifically expressed in the proliferating cells of the SVZ but not the VZ. The VZ is marked by the expression of the Otx1 gene. Later in development, Svet1 and Otx1 were expressed in subsets of cells of upper (II-IV) and deep (V-VI) layers, respectively. In the reeler cortex, where the layers are inverted, Svet1 and Otx1 label precursors of the upper and deeper layers, respectively, in their new location. Interestingly, in the Pax6/small eye mutant, Svet1 activity was abolished in the SVZ and in the upper part of the cortical plate while the Otx1 expression domain remained unchanged. Therefore, using Svet1 and Otx1 as cell-type-specific molecular markers for the upper and deep cortical layers we conclude that the Sey mutation affects predominantly the differentiation of the SVZ cells that fail to migrate into the cortical plate. The abnormality of the SVZ coincides with the absence of upper layer cells in the cortex. Taken together our data suggest that while the specification of deep cortical layers occurs in the ventricular zone, the SVZ is important for the proper specification of upper layers.

Animals↗
PubMed2000

The novel PCR-based technique of genotyping applied to identification of scrambler mutation in mice.

For a wide range of research purposes it is necessary to perform genotyping, i.e. to test which alleles, each corresponding to a particular locus, are present in the individual genome. Here we suggest a protocol of genotyping for mice with scrambler (scm) mutation. This mutation results in the aberrant splicing of the corresponding mRNA and affects the expression of mdab1 protein. Traditional approaches using genomic Southern hybridization or PCR with specific primers are not suitable for the genotyping of scm because of the lack of comprehensive information on the organization of the gene and on the presence of repetitive sequences in the known region. Here we propose a quick and highly reproducible method for genotyping scm mutant mice. The protocol consists of the following steps: isolation of genomic DNA, digestion with the restriction endonuclease, anchoring of resulting fragments with the adapter, and PCR amplification using adapter-specific primers. The final product of PCR amplification has a characteristic length which is different for the wt (wild type) and scm alleles. Thus, the characteristic pattern of bands obtained for each individual mouse specimen serves as criteria for the presence of wild type and/or scm allele. We believe that this approach could have wider application. The protocol can be easily modified and used as a convenient tool for identification of other genomic defects including those artificially introduced into genome by knockout or gene-trap techniques.

Animals↗
PubMed2000

Crystal structure of an RNA duplex containing phenyl-ribonucleotides, hydrophobic isosteres of the natural pyrimidines.

Chemically modified nucleotide analogs have gained widespread popularity for probing structure-function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 A resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3'-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs.

Base Sequence↗
PubMed2000

Pharmacokinetics and tolerability of an antiangiogenic ribozyme (ANGIOZYME) in healthy volunteers.

The pharmacokinetics and tolerability of a chemically stabilized synthetic ribozyme (ANGIOZYME) targeting the Flt-1 VEGF receptor mRNA were evaluated in healthy volunteers. In a placebo-controlled, single-dose escalation study, ribozyme was administered as a 4-hour i.v. infusion of 10 or 30 mg/m2 or as a s.c. bolus of 20 mg/m2. Peak ribozyme plasma concentrations of 1.5 and 3.8 micrograms/mL were observed after the 10 and 30 mg/m2 i.v. infusions, respectively. When normalized to dose, AUC values as well as peak concentrations increased proportionally as the dose was increased from 10 to 30 mg/m2. Peak concentrations of 0.9 microgram/mL were observed approximately 3.25 hours after a 20 mg/m2 s.c. bolus of ribozyme. The dose-normalized AUCs obtained after s.c. dosing were compared to the mean dose-normalized AUC after i.v. dosing to estimate an absolute s.c. bioavailability (f) of approximately 69%. An average elimination half-life of 28 to 40 minutes was observed after i.v. administration, which increased to 209 minutes after s.c. administration. Only 4 of 12 reported adverse events were possibly related to administration of ribozyme (headache and somnolence). Thus, ribozyme administration was well tolerated after a single 4-hour i.v. infusion of up to 30 mg/m2 or a single s.c. bolus of 20 mg/m2.

Adult↗
PubMed1998

Structural variation induced by different nucleotides at the cleavage site of the hammerhead ribozyme.

The hammerhead ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerhead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Watson-Crick base pair with C3 in the conserved core of the hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabilizing an inactive conformation of the molecule. Isotope-edited NMR experiments on the hammerhead complexes show that there are different short proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demonstrate that there are significant differences in the structure and/or dynamics of the active-site residues in these hammerhead complexes. Molecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the hammerhead ribozyme-substrate complexes are compared with the X-ray structure of the hammerhead ribozyme and are used to help understand the thermodynamic and kinetic differences among the cleavage-site variants.

Binding Sites↗
PubMed1998

The structural basis of hammerhead ribozyme self-cleavage.

We have captured an 8.7 A conformational change that takes place in the cleavage site of the hammerhead ribozyme during self-cleavage, using X-ray crystallography combined with physical and chemical trapping techniques. This rearrangement brings the hammerhead ribozyme from the ground state into a conformation that is poised to form the transition state geometry required for hammerhead RNA self-cleavage. Use of a 5'-C-methylated ribose adjacent to the cleavage site permits this ordinarily transient conformational change to be kinetically trapped and observed crystallographically after initiating the hammerhead ribozyme reaction in the crystal. Cleavage of the corresponding unmodified hammerhead ribozyme in the crystal under otherwise identical conditions is faster than in solution, indicating that we have indeed trapped a catalytically relevant intermediate form of this RNA enzyme.

Binding Sites↗
PubMed1998

Inductive interactions regulating body patterning in planarian, revealed by analysis of expression of novel gene scarf.

Subtractive hybridization was used to search for the early difference in gene expression between anteriorly and posteriorly regenerating tissues of the same region of the planarian body. A sequence (named scarf) specific for posteriorly regenerating tissue was isolated, encoding a soluble C-type lectin consisting of two slightly different carbohydrate-recognition domains. Such an unusual bivalent structure allows attribution of the protein to a novel subfamily of C-type lectins. scarf expression in intact planarian is rather abundant and follows a characteristic pattern not linked to any known morphological structure. We performed a series of experiments using scarf as a molecular marker. Its expression was monitored during different types of regeneration by whole-mount in situ hybridization and reverse-transcription polymerase chain reaction. The obtained data suggest that scarf expression is positively regulated by anterior tissues closely adjacent to the scarf-expressing region, so that their surgical removal results in rapid scarf switch-off. In turn, tissues expressing scarf seem to inhibit its activation anteriorly. This indicates that at least some of the body patterning events in planarians are based on a system of reciprocal inductive interactions rather than on a global morphogen gradient.

Amino Acid Sequence↗
PubMed1998

1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes.

The incorporation of 1-deazaadenosine (c1A, 1b) into a hammerhead ribozyme and the resulting catalytic activity is described. For this purpose the phosphoramidite 2a and the 3'-phosphonate 2b as well as Fractosil-linked 1-deazaadenosine (3b) were prepared. The methoxyacetyl group was used for the 6-amino group protection and the triisopropylsilyl residue was introduced as the 2'-OH protecting group. Replacement of residues A14and A15.1 of the hammerhead ribozyme by 1-deazaadenosine resulted in a significantly reduced catalytic activity. Substitution of the A6, A9 and A13 residues has only a minor influence. The findings observed on ribozymes modified with 1-deazaadenosine were compared with those containing other adenosine analogues.

Base Composition↗
PubMed1997

Ordered differential display: a simple method for systematic comparison of gene expression profiles.

A method for display of 3'-end restriction fragments of cDNAs is proposed, extending the idea reported recently. First, representative pools of such fragments are selectively amplified using PCR suppression effect. Then, simplified subsets of these fragments suitable for comparison by PAGE are amplified by adapter-specific primers extended by two randomly picked bases at their 3'ends. By testing all possible combinations of extended primers the whole mRNA pool may be systematically investigated. The method was applied to search for molecular regional markers of freshwater planarian Dugesia tigrina .

Animals↗
PubMed1997

Effect of structural modifications on the activity of the leadzyme.

The structure/function properties of functional groups in the leadzyme have been studied by assaying the activity of analog ribozymes generated by the systematic substitution of modified nucleotides in the internal loop region of the ribozyme. Guanosine analogs introduced at positions 4 and 7 occupied by guanosine in the wild-type molecule severely diminished cleavage. The substitution of deoxycytidine for cytidine at the cleavage site completely eliminated the activity of the leadzyme, as expected if the adjacent 2'-OH were the nucleophile in the cleavage reaction. On the other hand, substitution of an abasic nucleotide for adenosine at position 8 did not affect the activity of the ribozyme. An analysis of the activity of these analogs gives rise to the proposal of a triple-base pair motif implicating C1, G4, and G7.

Base Sequence↗
PubMed1997

Quantitating tertiary binding energies of 2' OH groups on the P1 duplex of the Tetrahymena ribozyme: intrinsic binding energy in an RNA enzyme.

Binding of the Tetrahymena ribozyme's oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme's internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2' OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2' OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using "substrate inhibition". This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2' OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to -2.9 kcal/mol. The energetic contributions of specific 2' OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A "threshold" model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G x U wobble at the cleavage site and the individual 2' OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energetic cost for docking the P1 duplex into the active site that is paid for by the "intrinsic binding energy" of groups on the P1 duplex. Substrates that lack sufficient tertiary binding energy to overcome this energetic barrier exhibit reduced reactivities. Thus, the ribozyme appears to use the intrinsic binding energy of groups on the P1 duplex for catalysis. This intrinsic binding energy may be used to position reactants within the active site and to induce electrostatic destabilization of the substrate, relative to its interactions in solution.

Animals↗
PubMed1997

Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect.

Here we describe a method for preparing high-quality cDNA libraries from total RNA. By this method, double-stranded (ds) cDNA ligated with a specially designed ds adaptor is amplified by PCR using a modified T-primer and another primer corresponding to the outer part of the adaptor. The suppression PCR effect strongly inhibits the amplification of poly(A) RNA, thereby reducing background. This method leads to amplification of high-quality cDNA, facilitating the construction of representative cDNA libraries from as little as 10-100 ng of total RNA.

DNA, Complementary↗