Ultrasound versus anatomical landmarks: Immediate complications in the central venous catheterization in children under 18 years of age. / Ultrasonido frente a referencias anatómicas: complicaciones inmediatas en la canalización de vía venosa central en menores de 18 años.

INTRODUCTION: The insertion of a central venous line in children and adolescents is technically more difficult, due to the smaller size of the structures. This can lead to an increase in immediate complications, which can be reduced when using ultrasound. In our institution, the percentage of these complications and the use of ultrasound are unknown. OBJECTIVE: To describe the frequency of immediate complications of central venous catheterisation guided by the ultrasound in a general university hospital, compared to the anatomical landmarks technique in children less than 18years of age. MATERIALS AND METHODS: Observational, retrospective, and analytical study, comparing the frequency of complications with two central venous catheterisation techniques: anatomical landmarks and ultrasound, according to the clinical records of procedures performed from June to November 2016. RESULTS: A total of 201 procedural records were analysed, of which 71% were with landmarks, and 29% with ultrasound. The overall incidence of immediate complications was 18.4%, with 12% using ultrasound and 21% using landmarks (OR: 0.5; 95%CI: 0.2-1.2). Children under 5years of age presented with 90% of the complications, the most frequent being the impossibility of passing the guide (29.7%) and multiple punctures (24.3%). There was no arterial puncture with use of ultrasound. Ultrasound was used by 13.4% of paediatric surgeons, by 32.4% of paediatricians, and 46.4% of anaesthetists, with complications of 25%, 19%, and 7%, respectively. The main indication for catheterisation was the need for vasoactive agents (74%), with the procedure being more complicated in patients with no peripheral venous accesses (46%). The success rate with anatomical landmarks was 77.6%, compared to 91.4% with ultrasound. CONCLUSION: Central venous catheterisation with ultrasound guidance in children under 18 reduces immediate complications by 42.8% and improves the success rate by 13.8%.

Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment.

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300µg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50µg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300µg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50µg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.

Tubulin carboxypeptidase assay based on the action of the enzyme on [14C]tyrosinated tubulin bound to nitrocellulose membrane.

We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification). Our method is simpler, less time-consuming, and suitable for multiple, simultaneous determinations and the substrate bound to nitrocellulose can be stored for several months without significant alteration of its properties. Peptidases other than tubulin carboxypeptidase can act on [14C]tyrosinated tubulin bound to nitrocellulose, solubilizing radioactive compounds, suggesting the eventual applicability of this method to assay proteases in general. Other features and advantages of the assay as well as its limitations are discussed.

Tubulin carboxypeptidase/microtubules association can be detected in the distal region of neural processes.

The association of tubulin carboxypeptidase with microtubules has been demonstrated in crude brain extracts and in living non-nervous cells. Here, we studied this phenomenon in cultured brain cells. To determine the association of the enzyme with neural microtubules we isolated the cytoskeletons (detergent-extraction under microtubule-stabilizing conditions) and measured the content of Tyr, Glu, and delta2 tubulin as a function of the in vitro incubation time of the cytoskeletons. The carboxypeptidase was found associated with microtubules in 2 days-cultured cells but not in 7 days-cultured cells. Quantitative analysis of digitized images after immunofluorescent staining revealed that detyrosination during the incubation of the cytoskeletons occurred preferentially in the distal regions of the neural processes. Prolonged taxol-treatment of the cells promoted higher detyrosination but Tyr tubulin was not depleted suggesting the existence of a subset of microtubules that has not associated carboxypeptidase and therefore cannot be detyrosinated even after prolonged taxol-treatment. This hypothesis was supported, although not conclusively, by additional experiments.

Incidence of intracranial bullet fragment migration.

Migration of retained bullets or bullet fragments may present as a complication of gunshot wounds to the head. This phenomenon has been reported in cases of abscess formation or retained copper fragments. Management of such migratory fragments is controversial. The purpose of this study is to determine the incidence of fragment migration in a population of neurosurgical patients treated for gunshot wounds to the head. Two-hundred and thirteen cases treated at Detroit Receiving Hospital between 1985 and 1987 were reviewed. Each patient treated had initial and one week follow-up imaging studies. Nine cases of documented migratory intracranial bullet fragments were identified. Thus, the incidence in this population is 4.2%. The fragments in eight cases were composed of copper, and in the remaining case, lead. No case was associated with an abscess. Fragments in the anterior fossa were found to migrate towards the sella turcica, while those of the middle fossa and posterior hemispheres migrate towards the confluence of sinuses (Torcula Herophili). Fragment migration was documented as early as 36 h post-injury. Based on this study, we recommend serial imaging studies to look for migrating bullet fragments and surgical removal aided by intra-operative ultrasound to localize the fragment when possible.

Association of tubulin carboxypeptidase with microtubules in living cells.

Tubulin carboxypeptidase is the enzyme that releases the C-terminal tyrosine from alpha-tubulin, converting tyrosine-terminated (Tyr) to detyrosinated (Glu) tubulin. The present study demonstrates that this enzyme is associated with microtubules in living cells. We extracted cultured cells (COS-7) with Triton X-100 under microtubule-stabilizing conditions and found tubulin carboxypeptidase activity in the cytoskeleton fraction. We ruled out, by using several control experiments, the possibility that this result was due to contamination of the isolated cytoskeletons by non-associated proteins contained in the detergent fraction or to an artifact in vitro during the extraction procedure. The associated carboxypeptidase activity showed characteristics similar to those of brain tubulin carboxypeptidase and different from those of pancreatic carboxypeptidase A. In comparison with cultures at confluence, those at low cell density contained small (if any) amounts of carboxypeptidase activity associated with microtubules. In addition, the enzyme was shown to be associated only with cold-labile microtubules. The tubulin carboxypeptidase/microtubule association was also demonstrated in Chinese hamster ovary, NIH 3T3 and PC12 cells. Interestingly, this association was not observed in cultured embryonic brain cells. Our results demonstrate that tubulin carboxypeptidase is indeed associated with microtubules in living cells. Furthermore, the findings that this association occurs with a subset of microtubules and that its magnitude depends on the degree of confluence of the cell culture indicate that it could be part of the mechanism that regulates the tyrosination state of microtubules.

The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes.

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the alpha-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75-78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.5 mM ATP during the in vitro microtubule formation resulted in a low recovery of carboxypeptidase activity in the microtubule fraction. This ATP-induced effect was not due to alteration of the enzyme activity or to inhibition of microtubule assembly but to a decrease of the association of the enzyme with microtubules. We found that the ATP-induced effect was not mediated by modifications on the microtubules but, presumably, on the enzyme molecule. The non-hydrolyzable ATP analogue, AMP-PCP, did not reproduce the effect of ATP. The inclusion of phosphatase inhibitors in the homogenization buffer also led to a decrease in the amount of tubulin carboxypeptidase associated with microtubules. Finally, we found that, in concordance with the mechanism hypothesized, the magnitude of the carboxypeptidase/microtubule association correlated well with the different incubation conditions created to favor maximal, minimal or intermediate protein phosphorylation states.

1;19 translocation in human meningioma.

BACKGROUND: Loss of chromosome 22 represents the most common chromosome abnormality (70%) in meningiomas. The remainder (30%) have a normal karyotype. Not only are the structural changes rare, they also occur simultaneously with various chromosome losses. METHODS: The authors identified and studied the meningiomas of two patients with standard tumor cell culture technique and chromosome preparation. RESULTS: Twenty karyotypes from each meningioma had a 46 modal chromosome number with t(1;19) (q21;p13) in all cells. CONCLUSIONS: The sole change of the (1;19) translocation in meningioma, without any other changes such as chromosome loss, as shown in this study, is unique and has never been reported before in the literature, to the knowledge of the authors. Additional study is needed to learn more about the rate of occurrence and the significant impact on meningeal tumor genesis.

Tyrosinatable and non-tyrosinatable tubulin subpopulations in rat muscle in comparison with those in brain.

Using immunobinding and enzymatic assays we determined in rat muscle extracts the proportion of tyrosinatable tubulin, that is, tubulin that participates in the tyrosination/detyrosination cycle. We found that in muscle, in contrast with nervous tissue, practically all tubulin molecules are tyrosinatable. In the case of rat brain the non-tyrosinatable tubulin pool accounts for about 50% of the tubulin. In addition, isolectrofocusing of 14C-tyrosinated tubulin from brain and muscle extracts revealed a different composition in tyrosinatable tubulin isotypes. One of the isotypes, which in muscle accounts for 86% of the 14C-tyrosinated tubulin species, was detyrosinated by the action of tubulin carboxypeptidase faster than the rest of the 14C-tyrosinated tubulin isotypes taken in whole. In the case of brain extract, that isotype accounts for only 16% of the labeled tubulin.

Recombinant granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for relapsed non-Hodgkin's lymphoma: blood and bone marrow progenitor growth studies. A phase II Eastern Cooperative Oncology Group Trial.

Sixteen patients with relapsed non-Hodgkin's lymphoma underwent autologous bone marrow transplantation and infusion of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Treatment consisted of involved-field radiotherapy, cyclophosphamide 60 mg/kg/d intravenously (IV) for 2 days, and fractionated total body irradiation (1,200 cGy). Autologous bone marrow was thawed and infused IV, followed 3 hours later by the first infusion of IV rhGM-CSF 11 micrograms/kg/d over 4 hours. Infusions of rhGM-CSF were continued daily until either both neutrophil count exceeded 1,500/microL and platelet count exceeded 50,000/microL, or until 30 days after marrow re-infusion. Toxicities encountered were mild and included fever, chills, hypertension, alopecia, rash, diarrhea, stomatitis, myalgias, and synovial (knee) effusions. Neutrophil recovery greater than 500/microL occurred a median of 14 days (range, 9 to 30 days) after marrow infusion, significantly earlier than in a comparable group of historic controls who recovered counts at a median time of 20 days (range, 12 to 51 days) (P = .00002). Median time to self-sustaining platelet counts greater than 20,000/microL was 23.5 days (range, 12 to 100 days), comparable with the historic group (P = .38). One bacteremia (central venous catheter exit site infection with Staphylococcus epidermidis) and one local infection (Giardia lamblia in stool) occurred. Patients received a median of 11.4 (range, 4.4 to 20.2) x 10(4) colony-forming unit granulocyte-macrophage (CFU-GM) progenitors per kg. Stem cell progenitors CFU-GM, CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM), and burst-forming unit-erythroid (BFU-E) were detected in the bone marrow as early as 7 days after marrow re-infusion, and increased in proportion to peripheral blood counts, but by 30 to 60 days still remained much lower than before transplant. Neutrophils transiently decreased in 13 of 16 patients (median decrease, 42%) within 24 to 72 hours of discontinuing rhGM-CSF infusions. These data suggest that rhGM-CSF therapy enhances neutrophil recovery by forcing stem cells to produce mature elements at an enhanced rate but may not affect marrow stem cell and early progenitor population sizes.

Effect of polyanions and polycations on detyrosination of tubulin and microtubules at steady state.

Microtubule protein preparations purified from rat brain were used to study the effect of polycations and polyanions on the release of the COOH-terminal tyrosine of the alpha-chain of tubulin catalyzed by tubulin carboxypeptidase. (1) Most of the polycations and polyanions tested, independently of the ionogenic group, inhibited the reaction in a concentration-dependent fashion. Under steady-state conditions, detyrosination of the microtubule pool was inhibited to the same degree as occurred with the non-assembled tubulin pool, except in the case of chondroitin sulphate. This compound inhibited detyrosination of the non-assembled tubulin pool, but not that of microtubules. (2) Heparin, the most potent inhibitor tested, produced the dissociation of the carboxypeptidase from microtubules. Many, but not all, of the other microtubule-associated polypeptides were also dissociated by heparin. (3) Polylysine counteracted the inhibitory and dissociating effects of heparin. (4) Heparin protected tubulin carboxypeptidase against inactivation. Our results and previous reports describing, in nervous tissue, the presence of proteoglycans, RNA and basic proteins that inhibit detyrosination, suggest that tubulin carboxypeptidase might be physiologically modulated by electrically charged macromolecules.

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes.

Chicken erythroid cells at different stages of maturation were incubated with [14C]tyrosine to analyze the incorporation of this amino acid into the COOH-terminus of alpha-tubulin. The incorporated radioactivity was determined in the microtubule and nonassembled tubulin pools. At all maturation stages, nonassembled tubulin was more labeled than microtubules. Microtubules were significantly labeled in proerythroblasts, labeled to a lesser extent in erythroblasts and not labeled at all in mature erythrocytes. We also studied the distribution of the tyrosinating and detyrosinating enzymes, tubulin:tyrosine ligase and tubulin carboxypeptidase, respectively, between the assembled and nonassembled tubulin fractions. Tubulin:tyrosine ligase behaved as a soluble entity at all maturation stages, whereas tubulin carboxypeptidase was found partially associated with microtubules in chicken proerythroblasts and completely soluble in mature erythrocytes. The marginal band of toad erythrocytes was examined by immunofluorescence using antibodies specific to tyrosinated and to detyrosinated tubulin. This marginal band which is mainly tyrosinated could be detyrosinated by exposure of these cells, previously permeabilized, to exogenously supplied tubulin carboxypeptidase. Toad erythrocytes contained soluble tubulin carboxypeptidase which showed an activity similar to that of chicken erythrocytes.

Relationship between the tyrosination state of tubulin and the activities of tubulin:tyrosine ligase and tubulin carboxypeptidase in rat muscle during development.

Tubulin can be post-translationally modified by the incorporation or the release of a tyrosine residue at the COOH-terminus of the alpha subunit. The present study demonstrates that rat muscle soluble preparations contain tubulin carboxypeptidase besides tubulin:tyrosine ligase. The state of tyrosination of tubulin and the activities of both the ligase and the carboxypeptidase were examined in rat muscle during development. The proportion of tyrosinated tubulin with respect to tyrosinable tubulin (tyrosinated plus detyrosinated tubulin) decreased from 83% (new-born rats) to 28% (adult rats) with the corresponding increase in detyrosinated tubulin. The activities of the enzymes decreased continuously and in a near parallel fashion during development. These results indicate that the changes in the tyrosination state of tubulin can not be explained merely by changes in the enzyme activities. We also compared the ability of rat muscle and brain [14C]tyrosinated tubulin to act as substrate of the carboxypeptidase. Muscle tubulin was found to be a less efficient substrate than brain tubulin.

Posttranslational tyrosination/detyrosination of tubulin.

Tubulin can be posttranslationally modified at the carboxyl terminus of the alpha-subunit by the addition or release of a tyrosine residue. These reactions involve two enzymes, tubulin: tyrosine ligase and tubulin carboxypeptidase. The tyrosine incorporation reaction has been described mainly in nervous tissue but it has also been found in a great variety of tissues and different species. Molecular aspects of the reactions catalyzed by these enzymes are at present well known, especially the reaction carried out by the ligase. Several lines of evidence indicate that assembled tubulin is the preferred substrate of the carboxypeptidase, whereas nonassembled tubulin is preferred by the ligase. Apparently this posttranslational modification does not affect the capacity of tubulin to form microtubules but it generates microtubules with different degrees of tyrosination. Variation in the content of the carboxyterminal tyrosine of alpha-tubulin as well as changes in the activity of the ligase and the carboxypeptidase are manifested during development. Changes in the cellular microtubular network modify the turnover of the carboxyterminal tyrosine of alpha-tubulin. Different subsets of microtubules with different degrees of tyrosination have been detected in interphase cells and during the mitotic cycle. Data from biochemical, immunological, and genetic studies have been compiled in this review; these are presented, with pertinent comments, with the hope of facilitating the comprehension of this particular aspect of the microtubule field.

Tubulin, but not microtubules, is the substrate for tubulin:tyrosine ligase in mature avian erythrocytes.

Chicken erythrocytes, which contain a marginal band of microtubules, were used to study the influence of the aggregation state of tubulin on the post-translational incorporation of tyrosine into the alpha-tubulin subunit. We found that the incorporation of [14C]tyrosine occurs almost exclusively into the nonassembled tubulin pool. The marginal band was practically not labeled. The low incorporation into microtubules was not due to the lack of tubulin with acceptor capacity since after cold-induced disassembly, an additional amount of [14C]tyrosine could be incorporated. 14C-Tyrosinated tubulin of the nonassembled pool could not be incorporated into microtubules of the marginal band after prolonged incubation at 37 degrees C or when the marginal band was regenerated after cold-induced depolymerization. In erythrocytes, tubulin:tyrosine ligase behaved as a soluble entity when the cells were lysed under microtubule-preserving conditions.

Tyrosination of microtubules and non-assembled tubulin in brain slices.

Brain slices were used to examine comparatively the incorporation of [14C]tyrosine into the C terminus of alpha-tubulin of the microtubule and non-assembled tubulin pools. We found that the incorporation of [14C]tyrosine from 5 min up to 60 min of incubation was higher in microtubules than in non-assembled tubulin. The possibility that this result was due to the activity of tubulin carboxypeptidase or tubulin:tyrosine ligase during the in vitro isolation of tubulin was discarded. We also found that tubulin:tyrosine ligase was mainly associated with microtubules when brain slices were homogenized under microtubule-preserving conditions. Conversely the enzyme behaved as a soluble entity when homogenization was performed under conditions that do not preserve microtubules. In addition, soluble tubulin:tyrosine ligase did not become sedimentable when in vitro conditions were changed to induce the formation of microtubules. The results presented in this work indicate the possibility that, in vivo, microtubules and not tubulin dimers are the major substrate for tubulin:tyrosine ligase. This is in contrast with previous findings from in vitro experiments, which showed a preference of the ligase for non-assembled tubulin.

Tyrosination-detyrosination of the COOH-terminus of alpha-tubulin in oocytes and embryos of Bufo arenarum.

Soluble tubulin from Bufo arenarum oocytes and early embryos was shown to be composed mainly of the non-tyrosinable species. The low proportion of tyrosinable tubulin was almost exclusively constituted by the tyrosinated form. Compared with oocytes and embryos, toad brain contained a higher proportion of tyrosinable tubulin constituted mainly by the non-tyrosinated form. Tubulin carboxypeptidase was detected in toad brain but not in oocytes and embryos.

Herniated thoracic disks.

Thoracic disc herniation is uncommon. An incidence of 0.25 to 0.75 per cent of protruded disks are in the thoracic region. A peak incidence is noted in the fourth decade with 75 per cent of the protruded disks occurring below T8. Pain is the most common initial symptom, present in 57 per cent of the cases, followed by sensory disturbances and motor involvement. By the time of diagnosis, 90 per cent of the patients have signs of spinal-cord compression. Although myelography has been considered the test of choice, 8 per cent false negative results and a correct preoperative diagnosis of 56 per cent has been reported. Now, with CT scanning with and without metrizamide, more accurate diagnoses can be achieved, even with cases in which myelography is negative. There has been a considerable improvement in the surgical treatment of herniated thoracic disks with over an 80 per cent rate of success for surgical approaches other than the posterior approach (decompressive laminectomy). An early and accurate diagnosis, coupled with improvement in the surgical approach, offers a much better prognosis for patients with thoracic disk herniation.

Thoracic disc herniation. Improved diagnosis with computed tomographic scanning and a review of the literature.

Thoracic disc herniation is uncommon. One of the main problems in the treatment of thoracic disc herniation has been the lack of accuracy of diagnostic tests. Now, with the use of computed tomographic scanning with and without metrizamide in the subarachnoid space, this accuracy has greatly improved. Computed tomography scanning can demonstrate the type and level of the lesion even when the myelographic study is negative. We have reviewed 280 cases; a peak incidence was noted in the fourth decade with 75% of the protruded discs occurring below T-8. Back pain was the most common presenting symptom followed by sensory disturbances. By the time of diagnosis, 70% of the patients had signs of spinal cord compression. A small group of patients could be identified that invariably had a good prognosis. They had a history of trauma, symptoms lasting less than a month, and soft disc herniation. Regarding the results of surgical treatment, there was a success rate ranging from 57% for decompressive laminectomy to over 80% for the posterolateral, lateral, and transthoracic approaches.

Release of C-terminal tyrosine from tubulin and microtubules at steady state.

Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase.