5-enol-Pyruvyl-Shikimate-3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties).

The shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays L. var Black Mexican Sweet) cells. Homogeneous enzyme preparations were obtained by a four-step procedure using ammonium sulfate fractionation, anion- and cation-exchange chromatography, and substrate elution from a cellulose phosphate column. The last step resulted in two well-separated activities of about the same molecular weight. A 2000- to 3000-fold purification, with an overall recovery of one-fourth of the initial activity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, activation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. Two EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. This allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throughout, whereas the other increased in exponentially growing cells and declined in late-logarithmic phase. The analysis of highly purified plastid preparations demonstrated a plastidial localization of both proteins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on defense-related aromatic biosynthesis in higher plants, are discussed.

Surgical needle design: torsion resistance.

OBJECTIVE: The interface between a surgical needle and forceps affects needle-forceps stability. Stability has become more important with the introduction of blunt-point surgical needles. The stability at the needle-forceps interface can be assessed by quantitating resistance to both rotation and twisting. Two commonly used surgical needles are the MO and CT needles, which are particularly useful for soft tissue closure for which significant strength of closure is required. Their comparison is thus of importance to surgeons. The design of the MO needle (greater flat section width than the CT needle) should allow for a greater resistance to rotation at the needle-forceps junction. This theoretically makes the MO needle a better choice when blunt-point needles (which require a greater force to penetrate tissue) are used. The objective of this study is to document and quantitate the differences, if any, between the CT and MO needles with regard to rotation and twisting at the needle-forceps junction. METHODS: To compare the efficacy of the CT and MO needles with regard to needle-forceps stability, the resistance of the needles to rotation and twisting was assessed using a Hios HP-10 digital torque meter. RESULTS: The resistance to twisting of the CT and MO needles was not significantly different. However, the MO needle is 25% more resistant to rotation than the CT needle. This differed by 10% from the expected difference. CONCLUSION: The resistance to twisting is not significantly affected by needle geometry, whereas the resistance to rotation is significantly affected. The difference between expected and observed differences in rotation is explained by study design.

Selection and Nuclear DNA Analysis of Cell Hybrids between Daucus carota and Oryza sativa.

In order to study the fate of the parental genomes in somatic cell hybrids between distantly related species, protoplasts from cultured cells of Daucus carota and Oryza sativa were fused. Selective conditions resulted, exclusively, in the growth of hybrid colonies which combined the capacity to multiply of carrot cells with the natural resistance to A2CA of rice cells. A methodology for measuring the relative contribution of the parental cells to the hybrid nuclear genome has been worked out. This is based both on hybridization of nuclear DNA bound to nitrocellulose filters (dot hybridization) with radioactively labelled nuclear DNA from one of the parents and on agarose gel fractionation of nuclear DNA digested with restriction endonucleases. The dot hybridization analysis, performed on one of the D. carota x O. sativa cell hybrids, showed that the major portion of the nuclear genome is homologous with the carrot partner, with rice contributing only a minor fraction, along with the selected resistance gene(s). The homology was confirmed after agarose gel fractionation of restriction endonuclease BamHI-digested nuclear DNA. Furthermore, strong homology at the level of gene expression between hybrid and carrot cells was shown by polyacrilamide gel electrophoresis of total soluble proteins.

Popliteal artery entrapment syndrome: diagnosis and management.

A variety of anatomical abnormalities may product an external compression of the popliteal artery, resulting in a thrombotic occlusion. Between 1983 and 1995, 9 patients presented a popliteal artery entrapment syndrome, bilateral in two cases. Ultrasonography, Angio-CT scan and MRI showed the abnormal structures and position of the popliteal artery on popliteal fossa. Arteriography detected an arterial compression in 6 limbs and occlusion in 4 limbs as well as an associated popliteal aneurysm in one patient and distal embolism in four. The abnormal medial head of the gastrocnemius muscle was resected in 8 limbs, while fibrous membranes were resected in 3 limbs. Autogenous vein replacement was performed in 4 cases of popliteal artery occlusion and following resection of a popliteal artery aneurysm in one case. All patients recovered. In the followup period, the occlusion of the arterial reconstruction occurred after 28 months in one patient, with a mild claudication. Noninvasive vascular imaging techniques may allow early diagnosis and management of PAES, avoiding popliteal arterial wall lesions and its occlusion, with a better outcome.

Ultraviolet inactivation kinetics of Escherichia coli and Yersinia pseudotuberculosis in annular reactors.

Terrorist threats have precipitated the need for information on the ultraviolet (UV) resistance of potential biothreat agents in food processing, such as Yersinia pestis. The objective of this study was to characterize the resistance of the Yersinia species to UV treatment using a single-lamp annular UV reactor. A novel method is proposed to measure the inactivation kinetics of Yersinia pseudotuberculosis, a surrogate of Y. pestis. This proposed method can overcome the disadvantages of the traditional collimated beam approach for liquids with high absorptive properties, such as liquid foods. As a reference, an inactivation rate of Escherichia coli K12 in caramel model solutions was measured first. Both first-order and series-event inactivation models were used to fit UV inactivation data. For the series-event model, an inactivation constant of k(SE)= 0.675 cm(2)/mJ and threshold n= 4 were obtained for E. coli K12 with the coefficient of determination R(2)= 0.987 and the standard deviation of log(10) reductions sigma(y)= 0.133. For Y. pseudotuberculosis, k(SE)= 0.984 cm(2)/mJ and n= 3 were obtained with R(2)= 0.972 and sigma(y)= 0.212. In contrast, for the first-order inactivation model, the first-order inactivation constant k(1)= 0.325 cm(2)/mJ with R(2)= 0.907 and sigma(y)= 0.354 was found for E. coli; and k(1)= 0.557 cm(2)/mJ with R(2)= 0.916 and sigma(y)= 0.402 was obtained for Y. pseudotuberculosis. Based on R(2), sigma(y), and the maximum absolute and relative errors, the series-event inactivation model describes the UV inactivation kinetics of Y. pseudotuberculosis and E. coli better than the first-order model. It is apparent that Y. pseudotuberculosis is less resistant to UV light than E. coli K12.

Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide.

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities from cell suspension cultures of Daucus carota were shown to copurify on (NH4)2SO4 fractionation, DEAE Sephadex and methotrexate-Sepharose affinity chromatography and to share approximately the same Mr(183 kDa and 185 kDa respectively) as judged by gel filtration on Sephacryl S-200.The copurified protein migrated as a single band on polyacrylamide gel electrophoresis under denaturing conditions.Both activities could be eluted from the same position of the native gel.Moreover, methotrexate-resistant cell lines which overproduce DHFR revealed to have a parallel higher level of TS. It is therefore proposed and discussed that in carrot, similarly to protozoa, TS and DHFR are present on a single bifunctional polypeptide of 58 kDa.

Protein synthesis in Bacillus subtilis: differential effect of potassium ions on in vitro peptide chain initiation and elongation.

The preparation and fractionation of a highly active and stable in vitro protein-synthesizing system from Bacillus subtilis is described. Potassium satisfied the requirement for a monovalent ion when the initiation factor-dependent binding of formyl-methionyl-transfer ribonucleic acid and synthesis of formyl-methionyl-puromycin were assayed, whereas it inhibited the reactions for polyphenylalanine synthesis. On the other hand, the ammonium ion satisfied the requirement for all assayed reactions. The in vitro experimental evidence suggested that potassium is an inhibitor of one or a few specific reactions involved in peptide chain elongation in B. subtilis.

Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization.

The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP(+) oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000±2 500, composed of identical subunits of 58 400±1 000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 µM and 2.2 µM, respectively and a turnover number of 4 750 or 1 500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.

A gamma-like DNA polymerase in spinach chloroplasts.

A DNA polymerase has been extracted from spinach chloroplasts and purified by chromatography on DEAE-cellulose and hydroxyapatite. A great similarity between the purified chloroplast polymerase and the mammalian mitochondrial DNA polymerase gamma was found by several criteria: preference for the synthetic primer-template (dT)12-18 . poly(rA), optimal requirement for Mn2+ (0.1-1.0 mM), KCl (100 mM) and pH (8-9), high relative molecular mass (approximately 105,000), resistance to aphidicolin and inhibition by N-ethylmaleimide. Some peculiar features of the chloroplast DNA polymerase have, however, been noticed. The mammalian DNA polymerase gamma has been suggested to be responsible for the replication of mitochondrial DNA. Thus, both the presence of a gamma-like DNA polymerase in chloroplasts and the similarities between the chloroplast and the mitochondrial DNA (absence of a nucleosomal structure an presence of displacement loops) lead to the suggestion that chloroplast DNA is also replicated by a gamma-like DNA polymerase and that the gamma polymerases present in eukaryotes are, therefore, involved in a strand-displacement DNA synthesis. An alpha-like DNA polymerase activity, present and predominant in crude leaf extracts, was practically absent from purified chloroplast preparations.

[Functional ano-rectal disorders: associated defecographic findings and related symptoms]. / Disordini funzionali ano-rettali: reperti defecografici associati e loro corrispondenza con la sintomatologia.

We retrospectively analyzed our latest 564 defecographies to evaluate: the frequency of each single defecographic finding on the total number of patients and in the two sexes; the frequency of the finding as single disorder and in association with other anorectal disorders; the correlation between findings and symptoms. The most common disorders were anterior rectocele (54%), mucosal prolapse (53%), puborectalis muscle syndrome (15%), and intussusception (15%); this order is respected in women (70, 57, 17 and 16% of the study population) but differs in men (mucosal prolapse 42%, puborectalis muscle syndrome 18%, posterior rectocele 14%, and intussusception 14%). Intussusception and puborectalis muscle syndrome were identified as an isolated finding in many cases (55% and 43%, respectively). The most common associations are: posterior rectocele with anterior rectocele (58% of cases), anterior rectocele with mucosal prolapse and vice versa (63% and 64%), intussusception with anterior rectocele (52%), puborectalis muscle syndrome with mucosal prolapse (36%) and with anterior rectocele (49%) and descending perineum syndrome with anterior rectocele (81%) and with mucosal prolapse (70%). The low rate of association among some disorders should be mentioned too for example, mucosal prolapse is associated, with intussusception only in 10% of cases. Clinical-defecographic correlations are less apparent: if some symptoms are found to be more typical of some disorders, this correlation is not pathognomonic and the clinical picture is quite aspecific. We stress the value of a careful correlation between clinical-anamnestic and defecographic findings for the correct evaluation of these disorders.

[Preoperative assessment of Blount disease]. / Valutazione pre-chirurgica del morbo di Blount.

Osteochondrosis of the medial proximal tibial epiphyseal plate is known as Blount disease. To date, two types have been described: infantile and juvenile disease. Six patients (5 men and 1 woman, mean age: 7.2 years) affected with infantile Blount disease diagnosed with plain radiography were submitted to Magnetic Resonance Imaging (MRI) for preoperative lesion staging. The lesions were bilateral in 5 patients. According to Langenskiöld classification, 2 patients were in stage II-III, 3 in stage III-IV and 1 in stage V-VI. Each patient underwent MRI of the more affected knee and 1 patient, who exhibited a more prominent metaphyseal beak, underwent CT of both knees. MRI depicted proximal tibia varus deformity and the degree of its angulation, which helped plan osteotomy; impaired growth of the medial portion of the proximal tibia involving physeal cartilage, metaphysis and epiphysis; alterations of menisci and ligaments (hypertrophic medial meniscus, medial collateral ligament laxity), the presence of bone bridges; the presence and integrity of unossified epiphysis and of chondral growth plate which was quite completely ossified in one case. To conclude, MRI is a completely atraumatic and noninvasive technique yielding many pieces of information necessary for the preoperative assessment of Blount disease.