3D average human corneal models.

In this paper we propose a method of building an average model or atlas of the cornea based on topographic data. Specific models can be constructed for the left or right eye, gender, age, or ametropia, to assess differences and similarities. An application of this atlas construction methodology to the study of corneal shape evolution with age is presented. Results show significant differences between age groups. This numerical atlas could also be helpful in the design of algorithms targeting the detection of corneal shape abnormalities, such as keratoconus or previous laser surgery.

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity.

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.

Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system.

The yeast LexA interaction trap was used to screen a cDNA library from Arabidopsis thaliana in order to identify proteins that interact with the viral protein genome linked (VPg)-proteinase of turnip mosaic potyvirus. The screen allowed the isolation of four candidate cDNA clones. Clones pHC4, pHC21, and pHC40 were partially sequenced but no homologies to known proteins were found. However, the amino acid sequence deduced from the complete nucleotide sequence of pSW56 revealed that it was the eukaryotic initiation factor (iso) 4E [eIF(iso)4E]. Deletion analysis indicated that the VPg domain was involved in the interaction with the plant protein. Interaction between the viral protein and the cellular protein was confirmed by ELISA-based binding experiments. eIF(iso)4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus.

Subgroup specific protection of mice from respiratory syncytial virus infection with peptides encompassing the amino acid region 174-187 from the G glycoprotein: the role of cysteinyl residues in protection.

We identified subgroup specific protective epitopes represented by the amino acid regions 174-187 and 171-187 of the G glycoproteins from respiratory syncytial virus (RSV), subgroups A and B. Mice immunized with coupled synthetic peptides corresponding to either the region 174-187 containing a Cys186-->Ser substitution or to the native region 171-187 were completely resistant to RSV infection but only to the respective virus. The protective activities of the peptides 174-187 were dependent on the Cys186-->Ser substitution. In addition, a recombinant protein representing the region 125-203 of the A subgroup G glycoprotein expressed in Escherichia coli was capable without further treatment to completely protect animals against RSV subgroup A infection. We show that the combinations of cysteinyl residues (positions 173, 176, 182, and 186) retained within either synthetic peptides or the recombinant protein G125-203 greatly influenced their protective activities. This indicates that the region 171-187 is essential for the protection conferred by the G125-203 protein. Furthermore, our results strongly suggest that the peptides' and recombinant protein's potencies are a function of a loop-like structure which is stabilized by intramolecular disulfide linkages between Cys176-Cys182 and Cys173-Cys186. This is further supported by the observation that chemical blocking of the sulfidryl groups in synthetic peptides completely eliminated their protective activity.

Transfection of murine fibroblast cells with human cytidine deaminase cDNA confers resistance to cytosine arabinoside.

One of the major limitations in the use of cytosine arabinoside (Ara-C) in cancer chemotherapy is the hematopoietic toxicity produced by this nucleoside analog. One approach to overcome this problem would be to insert a gene for drug resistance to Ara-C in normal hematopoietic cells to protect them from drug toxicity. An interesting candidate gene for this aim is cytidine deaminase which catalyzes the deamination of Arac-C, resulting in a significant loss of its antineoplastic activity. We have ligated the human cDNA for cytidine deaminase into the plasmid vector pMFG. Transfection of NIH 3T3-derived GP + E86 murine fibroblasts cells with this vector resulted in a marked increase (> 50-fold) in the expression of cytidine deaminase. In addition, the transfected cells showed resistance to the cytotoxic action and to the inhibition of DNA synthesis produced by Ara-C. Northern and Western blot analysis of the transfected cells showed increased expression of mRNA for cytidine deaminase and increased immunologically detectable enzyme. The ability to confer drug resistance to Ara-C through gene transfer of cytidine deaminase may have the potential as a selectable marker and for the protection of the bone marrow from the toxicity produced by this analog so as to increase its effectiveness in cancer chemotherapy.

Purification of turnip mosaic potyvirus viral protein genome-linked proteinase expressed in Escherichia coli and development of a quantitative assay for proteolytic activity.

The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH2, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPg-Pro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.

Evidence for an internal ribosome entry site within the 5' non-translated region of turnip mosaic potyvirus RNA.

The genomic RNA of potyviruses has a characteristic 5' non-translated region (5'NTR) to which a viral protein, VPg, is covalently attached. This suggests that the viral RNA lacks a conventional cap structure and thus its translation may not proceed in the same way as most cellular mRNAs. To investigate the role of the 5'NTR during translation, various derivatives of the turnip mosaic potyvirus (TuMV) leader were fused to the reporter gene beta-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit reticulocyte lysate and in planta following microprojectile DNA delivery into tobacco cell suspensions. GUS transcripts fused with the TuMV 5'NTR, whether they were capped or not, were efficiently translated, whereas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in excess over functional transcripts, translation was inhibited in a dose-dependent manner. Similarly, transcripts synthesized from the reverse complement of the 5'NTR inhibited translation to the same extent as the wild-type sequence, indicating that cap independence was not conferred by a specific sequence within the viral leader. A stable hairpin loop was placed in front or after the viral sequence. This hairpin loop normally prevented translation of control GUS transcripts but when the viral leader was positioned after it a significant level of GUS activity was measured, whether the transcripts were capped or not. On the other hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes bound to an internal site within the TuMV 5'NTR and then presumably scanned the sequence for the initiator AUG.

Human cytidine deaminase: purification of enzyme, cloning, and expression of its complementary DNA.

Cytidine (CR) deaminase was purified 47,000-fold to homogeneity from human placenta. The molecular mass of CR deaminase was estimated to be 48.7 kDa by gel filtration and 16.1 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it contains three or four identical subunits. We determined the amino acid sequence of several peptide fragments and designed 5'-primers to amplify, by the polymerase chain reaction, a specific 364-base pair DNA fragment using human liver complementary DNA (cDNA) as the template. This DNA fragment, which contains the codons of one peptide, was used as a probe to screen a cDNA library from human liver. We isolated and sequenced a cDNA clone of 910 base pairs that contained a 5' nontranslated region, a 438-base pair coding region, and a 3' nontranslated region with a polyadenylated tail. The translated region of the clone contained a deduced sequence of 146 amino acids, with a predicted molecular mass of 16.2 kDa and the sequences of our peptides. The cDNA was ligated in pGEX vector and expressed in Escherichia coli. The expressed protein had a high CR deaminase activity and molecular mass of 16.3 kDa. These data demonstrate clearly that the open reading frame of our cDNA clone codes for a functional human CR deaminase. Polymerase chain reaction amplifications of gene-specific DNA fragments from human/rodent hybrid cells indicate the localization of CR deaminase gene to human chromosome 1. The cDNA for CR deaminase will be a useful molecular probe to investigate the importance of this enzyme in chemotherapy.

Nucleic acid-binding properties of the P1 protein of turnip mosaic potyvirus produced in Escherichia coli.

The N-terminal P1 protein of turnip mosaic potyvirus (TuMV) polyprotein was overexpressed in Escherichia coli, purified by metal chelation chromatography under denaturing conditions and renatured. U.v. cross-linking experiments indicated that the recombinant protein interacted with RNA, and gel retardation electrophoresis demonstrated that more than one molecule of P1 bound one molecule of RNA. Formation of the protein-RNA complexes was dependent on the conformational state of P1 and was stable at relatively high concentrations of NaCl. P1 had the ability to bind ssRNA and ssDNA, with similar affinity, but was not able to bind to dsDNA. The TuMV protein had the additional characteristic of binding dsRNA with affinity similar to that observed with single-stranded nucleic acids.

Kinetic studies on 2',2'-difluorodeoxycytidine (Gemcitabine) with purified human deoxycytidine kinase and cytidine deaminase.

Phosphorylation of cytosine analogs by deoxycytidine kinase (dCK) and deamination by cytidine deaminase (CDA) are two important processes in the activation and elimination of these drugs. We have investigated the kinetic parameters of 2',2'-difluorodeoxycytidine (dFdC) using purified enzymes from human cells. Deoxycytidine (CdR) and dFdC had Km values of 1.5 and 4.6 microM for dCK, respectively. Feedback inhibition of dCK by deoxycytidine 5'-triphosphate (dCTP) was also studied. Our results show that dCTP produced a greater inhibition of the phosphorylation of dFdC than CdR with concentrations of dCTP ranging from 1 to 25 microM. dFdC was a good substrate for CDA. Kinetic studies with this enzyme gave Km values for CdR and dFdC of 46.3 and 95.7 microM, respectively. The effect of competitive inhibitors of CDA on the deamination of dFdC was also investigated. Diazepinone riboside was a more potent inhibitor than tetrahydrouridine using either CdR or dFdC as the substrate. Inhibitors of CDA could be useful in clinical trials in patients with cancer to increase the chemotherapeutic effectiveness of dFdC.

The complete nucleotide sequence of turnip mosaic potyvirus RNA.

The complete RNA genome of turnip mosaic potyvirus (TuMV) was amplified by seven consecutive reverse transcriptase-polymerase chain reactions and cloned into pUC9. The viral RNA is 9830 nucleotides long and contains a single open reading frame (ORF) of 9489 bases encoding a large polyprotein of 3863 amino acids with a calculated M(r) of 358,000. The non-coding region (NCR) preceding the ORF is 129 nucleotides long and has a high AU content (70%). Its predicted secondary structure is characterized by a hairpin loop with a free energy loss of -69.9 kJ/mol. The termination codon is followed by an AU-rich NCR of 209 bases, excluding the poly(A) tail. Seven potential nuclear inclusion a proteinase (NIa-Pro) recognition heptapeptides are found in the polyprotein. Their sequences agree with consensus potyviral NIa-Pro cleavage sequences except for that at the 6K-VPg site, which is characterized by a glutamic acid residue preceding the hydrolysed peptide bond. The TuMV proteins are similar to their corresponding potyviral proteins.

Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus in Escherichia coli.

The coding region for the precursor 6K-small nuclear inclusion a (NIa) protein and for the NIa protein of turnip mosaic potyvirus (TuMV) were introduced into the plasmid pET-11d for high-level expression in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses of E. coli proteins showed that the NIa protein underwent endoproteolysis and released a 22-kDa polypeptide. NH2-terminal amino acid sequencing of the recombinant 22-kDa protein was performed and was identical to the predicted amino end of the NIa protein. Site-directed mutagenesis confirmed that the hydrolysis was associated with the NIa proteolytic activity and that the proteinase recognized a Glu residue within an amino acid sequence found in the NIa protein which fitted the TuMV consensus cleavage site sequence. Fusion of the 6K protein with the NIa protein partially inhibited the hydrolytic reaction. The recombinant 22-kDa protein is likely the VPg of TuMV.

Cloning, characterization, and expression of a cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat.

We have isolated, sequenced, and expressed a cold-specific cDNA clone, Wcs120, that specifically hybridizes to a major mRNA species of approximately 1650 nucleotides from cold-acclimated wheat (Triticum aestivum L.). The accumulation of this mRNA was induced in less than 24 hours of cold treatment, and remained at a high steady-state level during the entire period of cold acclimation in the two freezing-tolerant genotypes of wheat tested. The expression of Wcs120 was transient in a less-tolerant genotype even though the genomic organization of the Wcs120 and the relative copy number were the same in the three genotypes. The mRNA level decreased rapidly during deacclimation and was not induced by heat shock, drought, or abscisic acid. The Wcs120 cDNA contains a long open reading frame encoding a protein of 390 amino acids. The encoded protein is boiling stable, highly hydrophilic, and has a compositional bias for glycine (26.7%), threonine (16.7%), and histidine (10.8%), although cysteine, phenylalanine, and tryptophan were absent. The WCS120 protein contains two repeated domains. Domain A has the consensus amino acid sequence GEKKGVMENIKEKLPGGHGDHQQ, which is repeated 6 times, whereas domain B has the sequence TGGTYGQQGHTGTT, which is repeated 11 times. The two domains were also found in barley dehydrins and rice abscisic acid-induced protein families. The expression of this cDNA in Escherichia coli, using the T(7) RNA polymerase promoter, produced a protein of 50 kilodaltons with an isoelectric point of 7.3, and this product comigrated with a major protein synthesized in vivo and in vitro during cold acclimation.

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants.

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.

Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2'-deoxycytidine by human cytidine deaminase.

Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues, inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine, 5-F-Zebularine, and diazepinone riboside on the deamination of CR, ARA-C, and 5-AZA-CdR using highly purified human CR deaminase (EC 3.5.4.5). These inhibitors produced a competitive type of inhibition with each substrate, the potency of which followed the patterns diazepinone riboside greater than 5-F-Zebularine and THU greater than Zebularine. 5-AZA-CdR was more sensitive than ARA-C to the inhibition produced by these deaminase inhibitors. The inhibition constants for diazepinone riboside lay in the range of 5-15 nM, suggesting that this inhibitor could be an excellent candidate for use in combination chemotherapy with either ARA-C or 5-AZA-CdR in patients with leukemia.

Sequence of the 3'-terminal region of turnip mosaic virus RNA and the capsid protein gene.

A sequence of 1801 nucleotides originating from the 3' end region of turnip mosaic virus (TuMV) RNA was cloned using the polymerase chain reaction and found to contain one long open reading frame (ORF). The amino acid sequence of three different regions of the isolated TuMV capsid protein (including the NH2 terminus) was determined and these partial sequences were found in the translation product predicted to be encoded by the large ORF. The data suggested that the TuMV capsid protein was a product arising from the maturation of a larger polyprotein, as observed for other potyviruses. Furthermore, the putative cleavage site corresponded to a glutamine-alanine dipeptide, a site commonly used in plant virus polyprotein processing. The capsid protein cistron was composed of 864 nucleotides and corresponded to a region encoding 288 amino acids with a calculated Mr of 33,186; the adjacent 3' non-coding region was 667 nucleotides long. The deduced amino acid sequence of the TuMV capsid protein is closely related to other potyvirus capsid proteins, with most of the variation being found within the NH2-terminal region.

[Laser angioplasty: a new approach in the treatment of iliac occlusion]. / L'angioplastie au laser: une nouvelle approache dans le traitement de l'occlusion iliaque.

Laser thermal angioplasty is a new technique. The longest follow-up is 3 years. It is used increasingly for the treatment of atheromatous disease of the femoropopliteal segment. The authors describe a case of successful recanalization of a completely occluded left external iliac artery using laser thermal angioplasty. The artery was approached through a left femoral dissection. The patient was discharged from the hospital on postoperative day 4 without claudication. The present indications and potential benefits and complications of this procedure are discussed.

Induction of cytidine deaminase in HL-60 myeloid leukemic cells by 5-aza-2'-deoxycytidine.

5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation and an active antileukemic agent, produced an induction of cytidine deaminase activity in human HL-60 myeloid leukemic cells. This increase in enzyme activity correlated with the induction of differentiation of the HL-60 leukemic cells as shown by an increase in nitroblue tetrazolium reduction, loss of clonogenicity and decrease in DNA synthesis. The concentration of 5-AZA-CdR that produced these effects was in the range of 0.1-1.0 microM. The increase in cytidine deaminase activity became apparent starting at 48 h from the start of the 5-AZA-CdR treatment and at 72 h was about 6-fold greater than the non-treated cell. Since cytidine deaminase is the major enzyme involved in the catabolism of cytosine nucleoside analogues, these results may be relevant in the evaluation of the clinical response of leukemic patients treated with 5-AZA-CdR.

Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A.

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.

Sequential hydrolysis of the gamma- and beta-phosphate groups of ATP by the ATP diphosphohydrolase from pig pancreas.

The ATP diphosphohydrolase (EC 3.6.1.5) from pig pancreas hydrolyzes triphospho- and diphosphonucleosides. The reaction products of ATP hydrolysis are ADP, AMP and orthophosphate, but AMP accumulates at a faster rate than ADP. A time-course study showed a simultaneous breakdown of ATP and ADP with initial rates for ATP and ADP hydrolysis of 2.1 and 3.8 mumol/min per mg protein, respectively. However, the rates reached similar values toward the end of the incubation period. According to double reciprocal plots and Dixon plots, the Km values for ATP and ADP are similar, Vmax for ADP hydrolysis is twice the Vmax for ATP hydrolysis and both nucleotides are competitive inhibitors of the other with their Ki values similar to their Km. These results are consistent with a sequential hydrolysis of the two diphosphoester bonds of ATP: ATP first binds to the enzyme, its gamma-phosphate group is hydrolyzed and released, resulting in an enzyme-ADP complex which either breaks down to free enzyme and ADP or is further processed via hydrolysis of the beta-phosphate group, releasing free enzyme, AMP and Pi. The experimental data showed that the processing step is favored.