DNA barcoding of the genus Lepidion (Gadiformes: Moridae) with recognition of Lepidion eques as a junior synonym of Lepidion lepidion.

DNA sequences of cytochrome c oxidase I gene (COI) from Lepidion spp. were employed to test the efficiency of species identification. A sample of 32 individuals from five Lepidion species was sequenced and combined with 26 sequences from other BOLD projects. As a result, 58 Lepidion DNA sequences of the COI gene belonging to eight of the nine recognized Lepidion species were analysed. Sequences were aligned and formed seven clades in a Bayesian phylogenetic tree, where Lepidion lepidion and Lepidion eques grouped jointly. The Kimura 2-parameter genetic distances, among congeners were, on average, 4.28%, 16 times greater than among conspecifics (0.27%). The main diagnostic meristic data of Lepidion spp. were compiled and a detailed morphological revision of the congeneric species L. eques and L. lepidion was made. The eye diameter was significantly different between L. eques and L. lepidion (P < 0.001). The number of anal fin rays ranged from 45 to 51 in L. lepidion and from 47 to 54 in L. eques, but no significant differences were obtained in the mean values of this variable (P = 0.07). According to the morphological and genetic analyses, the results strongly suggest that the Mediterranean codling L. lepidion and the North Atlantic codling L. eques are conspecific, making L. eques a junior synonym of L. lepidion.

Osteoblast-like cell response to macro- and micro-patterned carbon scaffolds obtained from the sea rush Juncus maritimus.

Carbon scaffolds with a directional patterned surface were obtained by pyrolysis of the sea rush Juncus maritimus. The structure of the scaffolds was investigated using scanning electron microscopy, mercury porosimetry and interferometric profilometry. X-ray diffraction and x-ray fluorescence were the techniques used for their chemical characterization. The alignment and differentiation of pre-osteoblasts (MC3T3-E1 cell line) incubated on the patterned scaffolds were evaluated by scanning electron microscopy, confocal laser scanning microscopy and by the quantification of the phosphatase alkaline activity and the osteocalcin synthesis. It was found that pyrolysis at 500 °C preserved and even enhanced the natural macro- and micro-patterning of the plant. The results obtained for porosity and chemical composition validated these structures as viable scaffolds for tissue engineering applications. Finally, the patterned surface was confirmed to promote the oriented growth of the pre-osteoblasts MC3T3-E1, not only after short periods of incubation (hours) but also after longer ones (several weeks). The quantification of the cell differentiation markers together with the evaluation of the cell layer morphology up to 28 days of incubation confirmed the differentiation of MC3T3-E1 cells to osteoblasts.

Three-dimensional bioactive glass implants fabricated by rapid prototyping based on CO(2) laser cladding.

Three-dimensional bioactive glass implants were produced by rapid prototyping based on laser cladding without using moulds. CO(2) laser radiation was employed to melt 45S5 and S520 bioactive glass particles and to deposit the material layer by layer following a desired geometry. Controlled thermal input and cooling rate by fine tuning of the processing parameters allowed the production of crack-free fully dense implants. Microstructural characterization revealed chemical composition stability, but crystallization during processing was extensive when 45S5 bioactive glass was used. Improved results were obtained using the S520 bioactive glass, which showed limited surface crystallization due to an expanded sintering window (the difference between the glass transition temperature and crystallization onset temperature). Ion release from the S520 implants in Tris buffer was similar to that of amorphous 45S5 bioactive glass prepared by casting in graphite moulds. Laser processed S520 scaffolds were not cytotoxic in vitro when osteoblast-like MC3T3-E1 cells were cultured with the dissolution products of the glasses; and the MC3T3-E1 cells attached and spread well when cultured on the surface of the materials.

Cytocompatibility of bio-inspired silicon carbide ceramics.

Due to its good mechanical and biochemical properties and, also, because of its unique interconnected porosity, bio-inspired silicon carbide (bioSiC) can be considered as a promising material for biomedical applications, including controlled drug delivery devices and tissue engineering scaffolds. This innovative material is produced by molten-Si infiltration of carbon templates, obtained by controlled pyrolysis of vegetable precursors. The final SiC ceramic presents a porous-interconnected microstructure that mimics the natural hierarchical structure of bone tissue and allows the internal growth of tissue, as well as favors angiogenesis. In the present work, the in vitro cytocompatibility of the bio-inspired SiC ceramics obtained, in this case, from the tree sapelli (Entandrophragma cylindricum) was evaluated. The attachment, spreading, cytoskeleton organization, proliferation, and mineralization of the preosteoblastic cell line MC3T3-E1 were analyzed for up to 28 days of incubation by scanning electron microscopy, interferometric profilometry, confocal laser scanning microscopy, MTT assay, as well as red alizarin staining and quantification. Cells seeded onto these ceramics were able to attach, spread, and proliferate properly with the maintenance of the typical preosteoblastic morphology throughout the time of culture. A certain level of mineralization on the surface of the sapelli-based SiC ceramics is observed. These results demonstrated the cytocompatibility of this porous and hierarchical material.

A new generation of bio-derived ceramic materials for medical applications.

A new generation of bio-derived ceramics can be developed as a base material for medical implants. Specific plant species are used as templates on which innovative transformation processes can modify the chemical composition maintaining the original biostructure. Building on the outstanding mechanical properties of the starting lignocellulosic templates, it is possible to develop lightweight and high-strength scaffolds for bone substitution. In vitro and in vivo experiments demonstrate the excellent biocompatibility of this new silicon carbide material (bioSiC) and how it gets colonized by the hosting bone tissue because of its unique interconnected hierarchic porosity, which opens the door to new biomedical applications.

In vitro testing of Nd:YAG laser processed calcium phosphate coatings.

Nd:YAG laser cladding is a new method for deposition of a calcium phosphate onto metallic surfaces of interest in implantology. The aim of this study was to compare the biologic response of MG-63 human osteoblast-like cells grown on Ti-6Al-4V substrates coated with a calcium phosphate layer applied using different methods: plasma spraying as reference material and Nd:YAG laser cladding as test material. Tissue culture polystyrene was used as negative control. The Nd:YAG laser clad material showed a behaviour similar to the reference material, plasma spray, respective to cell morphology (SEM observations), cell proliferation (AlamarBlue assay) and cytotoxicity of extracts (MTT assay). Proliferation, as measured by the AlamarBlue assay, showed little difference in the metabolic activity of the cells on the materials over an 18 day culture period. There were no significant differences in the cellular growth response on the test material when compared to the ones exhibited by the reference material. In the solvent extraction test all the extracts had some detrimental effect on cellular activity at 100% concentration, although cells incubated in the test material extract showed a proliferation rate similar to that of the reference material. To better understand the scope of these results it should be taken into account that the Nd:YAG clad coating has recently been developed. The fact that its in vitro performance is comparable to that produced by plasma spray, a material commercially available for more than ten years, indicates that this new laser based method could be of commercial interest in the near future.

Behaviour of MG-63 osteoblast-like cells on wood-based biomorphic SiC ceramics coated with bioactive glass.

The aim of this study was to test the in vitro cytotoxicity of wood-based biomorphic Silicon Carbide (SiC) ceramics coated with bioactive glass, using MG-63 human osteoblast-like cells, with a view to their application in bone implantology. To better understand the scope of this study, it should be taken into account that biomorphic SiC ceramics have only recently been developed and this innovative product has important properties such as interconnected porosity, high strength and toughness, and easy shaping. In the solvent extraction test, all the extracts had almost no effect on cellular activity even at 100% concentration, and cells incubated in the bioactive glass-coated SiC ceramics extracts showed a proliferation rate similar to that of the Thermanox control. There were no significant differences when the cellular attachment response of the cells on the wood-based biomorphic SiC ceramics, uncoated or coated with bioactive glass, was compared to the one exhibited by reference materials like Ti6Al4V and bulk bioactive glass. This fact looks very promising for biomedical applications.

Tratamiento endovascular de la coartación aórtica / Endovascular treatment of aortic coarctation

Introducción. El defecto congénito de la coartación de la aorta torácica se presenta en 1:10.000 habitantes, frecuentemente asociado a otros defectos congénitos del desarrollo cardíaco. Habitualmente se detecta y se repara en la infancia, y ocasionalmente se diagnostica en la adolescencia y la edad adulta, de novo o como complicación en la evolución de reparaciones previas (pseudoaneurismas, reestenosis, etc.). Estos pacientes requieren de intervenciones correctoras que se acompañan de una morbimortalidad significativa. La alternativa a ellas se ha desarrollado en los últimos años mediante técnicas endovasculares. Objetivo. Exponer el estado actual de la práctica clínica en el tratamiento endovascular de la coartación de la aorta torácica mediante angioplastia simple y angioplastia con stent. Desarrollo. Se discute la indicación de angioplastia en los niños menores de 3 años. La utilización de stents está limitada en la edad infantil por el desarrollo incompleto del niño. Puede ser el tratamiento de elección en el adolescente y el adulto, incluso mediante stent primario. Conclusión. Las reintervenciones con técnica endovascular de las reestenosis y las endoprótesis cubiertas para el tratamiento de los aneurismas poscoartación y de los pseudoaneurismas poscirugía ofrecen resultados satisfactorios

Introduction. Coarctation of the thoracic aorta presents as a connatal defect in 1:10,000 inhabitants, and is frequently associated to other congenital anomalies affecting the development of the heart. It is usually detected and repaired in infancy, but may be diagnosed during adolescence and in adulthood, either de novo or as a complication in the progress of previous repairs (pseudoaneurysms, restenosis, etc.). These patients require remedial interventions that are accompanied by a significant morbidity and mortality rate. An alternative to such procedures has been developed in recent years using endovascular techniques. Aim. To report the current state of the art of clinical practice in the endovascular treatment of coarctation of the thoracic aorta by means of simple stent angioplasty. Development. The indication for angioplasty in children under 3 years of age is discussed. The use of stents is limited in childhood because the child’s development is still incomplete. It can be the preferred treatment in adolescents and adults, even with the use of a primary stent. Conclusions. Reinterventions with endovascular techniques to treat restenosis and covered stents for the treatment of post-coarctation aneurysms and post-surgery pseudoaneurysms offer satisfactory results

Reparación endovascular de lesiones de aorta torácica descendente poscoartación / Endovascular repair of post-coarctation lesions in the descending thoracic aorta

Resumen. Objetivo. Presentamos dos casos de aneurisma de aorta torácica descendente (AATD) secundarios a coartación aórtica (CA) tratados mediante técnica endovascular. Casos clínicos. Caso 1: varón de 28 años intervenido de CA a los 3 meses de edad (coartectomía con anastomosis terminoterminal) y reintervenido a los 8 años (aortoplastia con parche de Dacron), que presenta una dilatación de 60 mm del área previamente intervenida y que interesa la salida de la arteria subclavia izquierda (ASI), con gradiente transaórtico de 21 mmHg. Requirió la realización de un bypass carotidosubclavio previo a la reparación aneurismática, implantando posteriormente dos endoprótesis solapadas tipo Talent de 26 × 130 mm y de 30 × 36 × 115 mm. Caso 2: mujer de 35 años, sin antecedentes personales de interés, con AAT descendente de 42 × 39 mm a 16 mm de la salida de la ASI, portadora de CA con gradiente transaórtico de 28 mmHg. Se implantó una endoprótesis aórtica tipo Talent de 32 × 130 mm con extremo proximal no recubierto en cayado aórtico proximal a ASI, sin oclusión de la salida de troncos supraaórticos, asociando una dilatación con balón de la zona residual de estenosis de la coartación. En ambos casos los AAT se excluyeron totalmente, sin fugas demostrables angiográfica ni topográficamente y sin gradientes transaórticos residuales. El resultado en el control clínico a 24 y 18 meses, los pacientes se encuentran asintomáticos, con el saco aneurismático trombosado y ausencia de fugas. Conclusión. Pese a que la respuesta del material y duración a largo plazo de las actuales endoprótesis se desconocen, el procedimiento endovascular puede convertirse en la técnica de elección en un número sustancial de pacientes con patología en aorta torácica descendente

Aims. We report on two cases of aneurysm of the descending thoracic aorta (DTA) secondary to aortic coarctation (AC) which were treated using endovascular techniques. Case reports. Case 1: a 28-year-old male who underwent an intervention to correct AC at the age of 3 months (coarctectomy with end-to-end anastomosis) and was submitted to surgery again at the age of 8 years (Dacron patch aortoplasty); there was a 60 mm dilatation of the area of the previous intervention with involvement of the exit point of the left subclavian artery (LSA), with a transaortic gradient of 21 mmHg. The patient required the placement of a carotid-subclavian bypass before aneurysmal repair, and later two overlapping 26 × 130 mm and 30 × 36 × 115 mm Talent-type stent grafts were implanted. Case 2: a 35-year-old female with no history of relevant clinical events, who had a 42 × 39 mm aneurysm of the DTA 16 mm from the exit point of the LSA; the patient was a carrier of AC with a transaortic gradient of 28 mmHg. A 32 × 130 mm Talenttype aortic stent graft with a non-covered proximal end was placed in the aortic arch proximal to the LSA, without occluding the exit of the supra-aortic trunks, and with the association of balloon dilatation of the stenotic region that remained from the coarctation. In both cases the aneurysms of the DTA were totally excluded, with no leaks that could be verified angiographically or topographically, and with no residual transaortic gradients. Results in the clinical control at 24 and 18 months showed that the patients are asymptomatic, with a thrombosed aneurysmal sac and absence of leaks. Conclusions. Despite the fact that the performance of the materials and the long-term duration of stent grafts in current use are unknown, the endovascular procedure may become the preferred technique in a substantial number of patients with a pathological condition affecting the descending thoracic aorta

Clinical significance of preoperative serum sialic acid levels in colorectal cancer: utility in the detection of patients at high risk of tumor recurrence.

This study was conducted to evaluate the significance of preoperative serum sialic acid levels in the diagnosis and prognosis of colorectal cancer (CRC). Total sialic acid (TSA) was determined by the thiobarbituric acid method and normalized to total protein (TP). A postoperative follow-up of CRC patients classified as Dukes' stages A, B or C was performed and survival analysis was carried out to evaluate the impact of sialic acid levels on tumor recurrence. Our diagnostic studies indicate that TSA/TP is a better marker than either TSA or carcinoembryonic antigen (CEA), especially for the detection of CRC patients at an early stage. At a cutoff of 30.90 nmol/mg of protein, TSA/TP showed a sensitivity of 85% with a specificity of 97% to discriminate CRC patients from healthy donors. In survival analysis, both TSA and TSA/TP were found to be significant prognostic factors for tumor recurrence in CRC. Furthermore, TSA/TP could distinguish patients at high risk of recurrence within Dukes' stage B and in multivariate analysis it was identified as the best independent prognostic factor. According to our results, preoperative serum TSA/TP content could supply additional information to that provided by Dukes' stage about the prognosis of CRC patients.

Calcium phosphate coatings obtained by Nd:YAG laser cladding: physicochemical and biologic properties.

The plasma spray (PS) technique is the most popular method commercially in use to produce calcium phosphate (CaP) coatings to promote fixation and osteointegration of the cementless prosthesis. Nevertheless, PS has some disadvantages, such as the poor coating-to-substrate adhesion, low mechanical strength, and brittleness of the coating. In order to overcome the drawbacks of plasma spraying, we introduce in this work a new method to apply a CaP coating on a Ti alloy using a well-known technique in the metallurgical field: laser surface cladding. The physicochemical characterization of the coatings has been carried out by means of X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). The biologic properties of the coatings have been assessed in vitro with human osteoblast-like MG-63 cells. The overall results of this study affirm that the Nd:YAG laser cladding technique is a promising method in the biomedical field.

Importancia de la hipercoagulabilidad en la cirugía de la isquemia crónica de extremidades inferiores / The importance of hypercoagulability in the surgery of chronic ischaemia of the lower limbs

Introducción. Se ha descrito una alta prevalencia de estados de hipercoagulabilidad en pacientes con isquemia crónica.Objetivo. Determinar la prevalencia e importancia de estados de hipercoagulabilidad en pacientes con patología oclusiva crónica de extremidades inferiores, que precisan revascularización. Pacientes y métodos. Estudio prospectivo octubre 1999-abril 2000. En 52 pacientes se determinó: antitrombina III, proteína C y S, anticuerpos anticardiolipina, plasminógeno, a2-antiplasmina y resistencia a la proteína C activada. Se registraron factores de riesgo, clínica, cirugía realizada y resultados, y se analizó su relación con alteraciones de hipercoagulabilidad. Resultados. El 6 por ciento presentaban déficit de antitrombina III; el 31 por ciento, déficit de proteína C; el 2 por ciento, déficit de proteína S; el 10 por ciento, anticuerpos anticardiolipina, y el 12 por ciento, resistencia a la proteína C activada (RPCA). El 29 por ciento de los pacientes presentaban una alteración; el 13 por ciento, más de una, y el 58 por ciento, ninguna. El 50 por ciento (3/6) de los pacientes con RPCA se trombosaron, frente al 13 por ciento (6/46) de los pacientes sin RPCA (p= 0,05). El 42 por ciento (3/7) de los pacientes con varias alteraciones se trombosaron, frente al 13 por ciento (6/45) del resto (p= 0,08). El 33 por ciento (2/6) de los pacientes con RPCA presentaron trombosis precoz, frente al 2,1 por ciento (1/46) del resto (p= 0,03). El 28 por ciento (2/7) de los pacientes con varias alteraciones presentaron trombosis precoz, frente al 2,2 por ciento (1/ 45) del resto (p= 0,04). Conclusiones. La prevalencia de estados de hipercoagulabilidad en isquémicos crónicos es elevada. Estos hallazgos inducen implicaciones terapéuticas. (AU)

Cell surface human alpha-L-fucosidase.

The acid alpha-L-fucosidase is usually found as a soluble component of lysosomes where fucoglycoconjugates are degraded. In the present investigation, we have demonstrated the existence of a cell surface protein with enzymatic alpha-L-fucosidase activity that crossreacts specifically with a rabbit anti-(alpha-L-fucosidase) Ig. By different approaches, this alpha-L-fucosidase, which represents 10-20% of the total cellular fucosidase activity, was detected in all the tested human cells (hemopoietic, epithelial, mesenchymal). Two bands of approximately 43-49 kDa were observed, although theoretical data support the possibility of having the same genetic origin that the known 50 to 55-kDa Mr alpha-L-fucosidase. We speculate about an alternative traffic pathway for the plasma membrane alpha-L-fucosidase to work on the rapid turnover of glycoproteins.

Value of the serum alpha-L-fucosidase activity in the diagnosis of colorectal cancer.

OBJECTIVES: The purpose of this study was to assess the value of the serum levels of alpha-L-fucosidase activity in the diagnosis of patients with colorectal cancer. METHODS: Using a fluorometric method we analyzed the alpha-L-fucosidase activity in preoperative sera from 137 colorectal cancer patients and in sera from 232 donors. RESULTS: The enzymatic activity of alpha-L-fucosidase was significantly lower (p <0.001) in patients (4.8+/-3.09 U/ml) than in donors (10.5+/-5.46 U/ml). Using the ROC curve, the ideal cut-off for the diagnostic value of alpha-L-fucosidase activity was determined to be 5.6 U/ml. The diagnostic efficiency for colorectal cancer of alpha-L-fucosidase activity was higher than that observed for carcinoembryonic antigen (cut-off 5.0 ng/ml), especially for tumors at an early stage. CONCLUSIONS: Our results suggest that preoperative serum alpha-L-fucosidase activity may be used as a cheap and easy complementary test, in addition to standard clinical procedures routinely used for the diagnosis of colorectal cancer.

Alpha-L-fucosidase enzyme in the prediction of colorectal cancer patients at high risk of tumor recurrence.

The aim of this study was to examine the prognostic value of the alpha-L-fucosidase enzyme to determine whether it can help in the early recognition of colorectal cancer cases at high risk of tumor recurrence. One hundred and twenty-three colorectal carcinoma patients treated by curative surgery were studied. The alpha-L-fucosidase activity was assayed in the tumor and in normal mucosa from each patient using a fluorometric method. Seven other clinical and pathologic features were also studied. To evaluate the impact of each variable over the disease-free interval, a postoperative 30-month follow-up of patients was performed, and a statistical survival analysis was carried out. The recurrence appearance was higher when the relative decrease of alpha-L-fucosidase activity was more than 52% (log-rank test, P = .0261). The results of this work indicate that alpha-L-fucosidase activity appears to be a good independent prognostic factor of tumoral recurrence in colorectal carcinoma.

Immunohistochemical analysis of sialic acid and fucose composition in human colorectal adenocarcinoma.

The expression of different sialoglycoconjugates and fucoglycoconjugates in normal mucosa and adenocarcinoma samples from 43 colorectal cancer patients was investigated by using specific lectins and applying a semiquantitative analysis. A pronounced decrease in the intracellular binding of the Maackia amurensis lectin, which recognizes alpha(2,3)-linked sialic acid residues, was found in the tumoral tissue. In contrast, a significant increase in the staining with the Sambucus nigra lectin (SNA I), which binds to alpha(2,6)-linked sialic acid residues, was detected in the epithelial cells as well as in the mucins from tumors. No significant differences in the reactivity with the Aleuria aurantia lectin, which recognizes the sequence Fuc(alpha1,6)GlcNAc, between normal and malignant colorectal tissues were detected. Furthermore, the correlation between lectin-binding profiles and the prognosis of colorectal cancer patients was examined. After an average postoperative follow-up period of 31 months, patients with tumors showing a strong SNA I staining presented a greater probability of disease recurrence. This result suggests that the intensity of staining with SNA I could be a valid parameter for predicting recurrence in colorectal cancer.

Nonradioactive immunoquantification of alpha-L-fucosidase protein in human colon tissues.

alpha-L-Fucosidase is a glycosidase involved in the degradation of fucoglycoconjugates and has a diagnostic significance because it has been described to be altered in several known diseases. However, in vitro studies on enzymatic activities may not reflect the real protein levels in tissues. This paper describes a simple method to quantify alpha-L-fucosidase protein levels in human crude extracts, combining the slot-blot technique and a nonradioactive immunoassay. Taking advantage of the similarities in different mammalian fucosidases, a polyclonal antiserum was raised against commercial purified alpha-L-fucosidase from bovine kidney that cross-reacted with the human colon enzyme. The method is able to detect as little as 0.75 ng alpha-L-fucosidase. To illustrate the direct application of this technique, we analysed and quantified alpha-L-fucosidase protein levels in 18 human colon crude samples. This technique could prove useful in clinical pathology, allowing fast and accurate measurement of alpha-L-fucosidase in crude extracts.

Isolation and characterization of mutants of vaccinia virus with a modified 94-kDa inclusion protein.

We have characterized one of the most highly expressed genes of vaccinia virus, WR strain, in the wild type and in several spontaneous mutants isolated from persistently infected cells. This gene encodes the 94-kDa inclusion protein, which is the vaccinia virus counterpart of the 160-kDa A-type inclusion (ATI) protein of cowpox virus. The homology index between both genes is greater than 95%. A deletion of two consecutive adenylate residues is responsible for a frameshift mutation and premature translational termination in the vaccinia virus gene. In addition, several point mutations and small deletions occur in the 94K gene. The deduced protein contains 725 amino acids, and 4 of the 10 repeated motifs present in the carboxyl terminus of the cowpox virus 160-kDa protein are conserved. In several mutants independently isolated from untreated and interferon-treated persistently infected cells, the gene encodes a 40-kDa protein. In mutant 87-4, this truncated protein is due to the insertion of a cytidilate residue that produces a frameshift mutation and premature translational termination. The deduced protein contains 366 amino acids and has lost all the repetitions. Transcriptional analysis has shown that the steady-state levels of mRNAs in cells infected with the mutants or wild-type vaccinia virus are similar. However, the accumulation of this protein in cells infected with the mutants is reduced indicating some instability. In addition the mutated protein is not recognized by polyclonal antisera. Existence of tandemly repeated sequences at the carboxyl terminus of this family of inclusion proteins correlates with their antigenicity. These results indicate a high degree of mutability of the ATI gene and products, which apparently has no consequence on replication in vitro, but could have relevance to control of the infection by immune responses in animal hosts.

Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae.

A vaccinia virus open reading frame (ORF) previously predicted to encode thymidylate kinase (TmpK) is shown to encode an active enzyme. A copy of the ORF, generated by polymerase chain reaction, was cloned into an Escherichia coli inducible expression vector. Cell extracts of E. coli expressing the vaccinia gene contained high levels of TmpK activity, whereas extracts of cells without the TmpK gene did not. The vaccinia ORF expressed from a yeast vector complemented a Saccharomyces cerevisiae cdc8 mutant, demonstrating functional compatibility of the vaccinia virus and yeast TmpK enzymes. The gene is shown to be nonessential for the replication of vaccinia virus in cultured cells by the construction of a viable virus mutant that has the coding region of the TmpK gene interrupted by the Ecogpt gene. Synthesis of the vaccinia TmpK protein in infected cells was demonstrated by the use of a polyvalent rabbit antiserum raised against the purified TmpK enzyme expressed in E. coli to immunoprecipitate a 23-kDa early polypeptide from cells infected with wild type vaccinia but not from cells infected with the TmpK mutant. Plasmid vectors that allow the construction of recombinant viruses expressing foreign gene(s) from the nonessential TmpK locus are described.

Vaccinia virus encodes a thymidylate kinase gene: sequence and transcriptional mapping.

The nucleotide sequence and deduced amino acid sequence of a vaccinia virus gene from the SalI F fragment are shown. The predicted polypeptide shares 42% amino acid identity over a 200 amino acid region with Saccharomyces cerevisiae thymidylate kinase (TmpK) and has low homology with herpes simplex virus deoxypyrimidine kinase. Northern blotting and S1 nuclease protection showed that the TmpK gene is transcribed early during infection and mapped the mRNA 5' end to immediately upstream of the second inframe ATG codon of the open reading frame (ORF). The encoded polypeptide is predicted to be 204 amino acids long (23.2 kD) and is almost colinear with yeast TmpK. Vaccinia virus possesses genes for TK and TmpK, separated by 57 kilobases of DNA, which are co-ordinately expressed and the encoded enzymes perform sequential steps in the same biochemical pathway.