Prognosis of deep infection in spinal surgery using implants, treated by retention, removal of bone graft and lengthy antibiotherapy. / Pronóstico de la infección profunda en la cirugía raquídea con implante, tratada mediante retención, retirada del injerto óseo y antibioterapia prolongada.

OBJECTIVE: Surgical site infections (SSIs) are complications that predispose to a high risk of unfavourable surgical outcomes. The aim of this study was to assess the SSI rate in this type of patients and their prognosis with similar treatment. MATERIALS AND METHODS: A retrospective case series of 799 patients above 18 years old with spinal instrumentation surgery, between January 2010 and December 2014 in the traumatology and orthopaedic surgery department of our institution. All patients with SSIs were treated by debridement, graft replacement, retention of the instrumentation and lengthy courses of antimicrobial therapy. The patients were followed up for a period of 12 months. RESULTS: Of all the patients with arthrodesis, 32 (4%) had spinal SSIs. Three patients were lost to follow-up. The final sample analyzed comprised 29 cases, with a median age of 54.9 years (IQR, 45.7-67 years) and a Charlson comorbidity index of 2.0 (IQR; 0-3). A microbiological diagnosis was obtained in 75.8% of the cases. Of these, the ISSs were monomicrobial in 68.2% and polymicrobial in 31.8%. Once treatment had been completed, 96% were cured without sequelae, and the rate of recurrence and reoperation was 4%. CONCLUSIONS: Treatment based on debridement, retention of the instrumentation, graft replacement and lengthy courses of antimicrobial therapy seems a very effective strategy in the treatment of patients with deep surgical site infection in spine surgery.

A Monte Carlo Study of the Photon Spectrum due to the Different Materials Used in the Construction of Flattening Filters of LINAC.

Different types the spectrum of photons were studied; they were emitted from the flattening filter of a LINAC Varian 2100 C/D that operates at 15 MV. The simplified geometry of the LINAC head was calculated using the MCNPX code based on the studies of the materials of the flattening filter, namely, SST, W, Pb, Fe, Ta, Al, and Cu. These materials were replaced in the flattening filter to calculate the photon spectra at the output of this device to obtain the spectrum that makes an impact with the patient. The different spectra obtained were analyzed and compared to the emission from the original spectra configuration of the LINAC, which uses material W. In the study, different combinations of materials were considered in order to establish differences between the use of different materials and the original material, with the objective of establishing advantages and disadvantages from a clinical standpoint.

Hysteroscopic myomectomy: our experience and review.

OBJECTIVE: To analyze the results of hysteroscopic myomectomy in our center and to compare the results to those published in the literature. METHODS: We performed a retrospective study of the clinical histories of patients who had undergone hysteroscopic myomectomy with a resectoscope between January 1992 and December 1999. Procedures were performed at a hysteroscopic clinic in the Department of Obstetrics and Gynecology at the University Public Hospital in Madrid's south zone. One hundred twenty pre-, peri-, and postmenopausal women with submucous myomas were included in the study. All patients underwent hysteroscopic resection with a monopolar loop. RESULTS: We performed 120 hysteroscopic myomectomies. The patients' median age was 44.8 years (23 to 74). Abnormal uterine bleeding (AUB) was the most frequent indication (84.1%). Inability to reproduce was the indication in 14 (11.6%) cases. GnRH analogue preparation was used in 60% of cases. We operated on 52 (43.3%) type 0, 51 (42.5%) type I, and 17 (14.1%) type II myomas, according to Wamsteker and Blok classification. A median of 32.5 (10 to 105) minutes was required for the interventions. The myomectomy was combined with another operation (12 polypectomies, 24 endometrial resections, and 1 laparoscopic ovarian cystectomy) in 32 patients. The median retention of glycemia was 281 cc (0 to 1300). We could not complete the resection in 22 patients. Twelve underwent reoperation (3 hysterectomies and 9 second myomectomies). No serious complications occurred, and the median hospital stay was 25.4 hours. The histological study confirmed leiomyoma in all the cases. The intervention results were satisfactory after a follow-up period of 12 months to 7 years, AUB being controlled in 88.5% of the patients. CONCLUSION: Hysteroscopic myomectomy is a reliable procedure that is effective in controlling abnormal uterine bleeding. It is a good alternative to hysterectomy and has an acceptable surgical time and minimum hospital stay. To reduce the need of reintervention, appropriate patient selection and improved technique are necessary. The technique also offers significant economic savings compared with the conventional surgical methods.

A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates, casein and calmodulin.

Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, alpha-subunits, and two regulatory, beta-subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is downregulated by the regulatory subunit. The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme. Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex. The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit. The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex. The interactions with the regulatory subunit and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30 degrees C. The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme. The results obtained suggest that CK2beta subunit and protein substrates share a common, or at least an overlapping, site of interaction on the catalytic subunit. The interaction between both subunits would prevent substrates from binding irreversibly to alpha subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate. The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine.

Severe menorrhagia due to Glanzmann thrombasthenia treated with hydrothermal ablation.

Glanzmann thrombasthenia is a rare platelet disorder inherited as an autosomal recessive trait. Abnormal uterine bleeding is a common problem in women with the disease. Medical management may not always be effective and further treatment may be necessary. Two women underwent endometrial ablation with a continuous-flow circulating hydrothermal ablator. After follow-up of 12 and 18 months, both women remained without abnormal uterine bleeding.

Binding of polylysine to protein kinase CK2, measured by Surface Plasmon Resonance.

The interaction between protein kinase CK2 and polylysine has been studied by Surface Plasmon Resonance (SPR). The binding process has a very low energy of activation, it is irreversible, and too slow as to explain the enzyme activity stimulation as a direct consequence of the polylysine binding. The polylysine interaction with a peptide substrate and with casein are faster, and in agreement with a substrate-mediated mechanism of activity stimulation. After several hours of incubation, the binding of polylysine to CK2 produces the loss of enzymatic activity.

Distribution of CK2, its substrate MAP1B and phosphatases in neuronal cells.

Neuronal morphogenesis depends on the organization of cytoskeletal elements among which microtubules play a very important role. The organization of microtubules is controlled by the presence of microtubule-associated proteins (MAPs), the activity of which is modulated by phosphorylation and dephosphorylation. One of these MAPs is MAP1B, which is very abundant within growing axons of developing neurons where it is found phosphorylated by several protein kinases including CK2. The expression of MAP1B is notably decreased after neuronal maturation in parallel with a change in the localization of the protein, which becomes largely concentrated in neuronal cell bodies and dendrites. Interestingly, MAP1B remains highly phosphorylated at sites targeted by protein kinase CK2 in mature neurons. We have analyzed the expression and localization of CK2 catalytic subunits along neuronal development. CK2alpha subunit appears early during development whereas CK2alpha' subunit appears within mature neurons at the time of dendrite maturation and synaptogenesis, in parallel with the change in the localization of MAP1B. CK2alpha subunit is found associated with microtubule preparations obtained from either grey matter or white matter from adult bovine brain, whereas CK2alpha' subunit is highly enriched in microtubules obtained from grey matter. These results lend support to the hypothesis that CK2alpha' subunit is concentrated in neuronal cell bodies and dendrites, where it associates with microtubules, thus contributing to the increased phosphorylation of MAP1B in this localization in mature neurons.

A surface-plasmon-resonance analysis of polylysine interactions with a peptide substrate of protein kinase CK2 and with the enzyme.

The mechanism of protein kinase CK2 (CK2) activity stimulation by polylysine has been studied by surface plasmon resonance (SPR). The kinetics of the polylysine interaction with a peptide substrate of the enzyme, and with the enzyme itself, have been investigated. A peptide containing a threonine (T) residue surrounded by a cluster of negatively charged acidic [arginine (R) and glutamic acid (E)] residues, RRREEETEEE, and specifically phosphorylated by CK2, was selected. Polylysine interacts with both the enzyme and the peptide substrate. The rate constant, the stoichiometry of the polylysine-peptide substrate interaction and the kinetic parameters of the stimulated enzyme were used to calculate the polylysine-dependent stimulation of CK2. The results are in agreement with experimentally determined polylysine-dependent stimulation. The polylysine-enzyme interaction is too slow to account for enzyme stimulation. The behaviour of polylysine is not reproduced by the polyamine spermine. The results are consistent with a substrate-mediated mechanism of CK2 stimulation by polylysine, and they suggest that the CK2 stimulation by polyamines occurs by a different mechanism.

[Risk of contamination with blood during obstetric-gynecologic surgery]. / Riesgo de contaminación con sangre durante cirugía gineco-obstétrica.

To determine the risk of exposure to blood and body fluids potentially contaminated with infectious organisms, we instituted a prospective study of 100 gynecologic procedures performed at Hospital General Regional of Puebla, México. Accidental exposure to blood occurred during 8 procedures (8%). There were 8 glove tears (8%). Needlestick injuries occurred in 6% of the operations. The frequency of blood contamination, glove tears and percutaneous injuries is high; surgical personnel are at risk of contracting a blood-borne disease such as HIV infection or viral hepatitis. Implantation of universal blood and body fluids precautions is useful in preventing HIV exposures of which needlestick precautions are most important.

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions.

Mg2+ as well as Mn2+, and Co2+, which may substitute Mg2+ in the mental ion requirement of casein kinase 2 (Gatica et al., FEBS Lett: 315:173-173, 1993), have been repeatedly reported to display an optimal concentration at which activity of casein kinase 2 is maximal. As far as we know this intriguing property has always been observed with casein as substrate. This phosphoprotein is not the natural substrate of the enzyme, and it is well known that it binds divalent metal ions, which provoke the aggregation and precipitation of the protein. Since an optimal concentration of metal ion might have a regulatory role, we have examined if it is a consequence of the particular properties of casein, or it is an inherent property of the enzyme, extensive to other substrates. We have used the type II regulatory subunit of protein kinase A which is a physiological substrate of the enzyme, and the peptide RRREEETEEE as a specific substrate. No optimal concentration of Mg2+ is observed when these two substrates are used. The results explain, however, why that optimum is observed with casein. Although low concentration of Mn2+, and Co2+ render about 25% of the maximal activity found with Mg2+, they inactivate the enzyme almost fully at concentrations at which Mg2+ yield the maximal activity.

A polylysine-induced aggregation of substrate accompanies the stimulation of casein kinase II by polylysine.

Casein kinase II (CK-II) activation by polylysine parallels an aggregation of substrates promoted by the polycation. CK-II is known to be stimulated by basic polypeptides and polyamines. The mechanism by which this stimulation takes place, however, is not yet fully understood. Here we show that, in the usual CK-II assay, polylysine induces the aggregation of casein. This aggregation has been monitored by turbidimetry, electron microscopy and gel filtration. The polylysine-concentration-dependence of the casein aggregation parallels the polylysine-concentration-dependence of the enzyme stimulation. In the presence of polylysine the enzyme is incorporated into the casein aggregates promoted by the polycation, thus supporting the view that this substrate aggregation is directly related to the mechanism of CK-II stimulation. Preliminary results show that a similar parallelism occurs with other natural substrates of the enzyme. The physiological meaning of this substrate aggregation, and its possible relation to other polylysine-stimulated enzymes and polylysine-aggregated proteins, are discussed.

Dipyridamole stimulates types II cAMP-dependent protein kinase in vitro.

Dipyridamole activates in vitro type II cAMP-dependent protein kinase. This agent stimulates the autophosphorylation of the regulatory subunit in the presence of cAMP but not so in the absence of the cyclic nucleotide. The activation was also observed with exogenous substrates such as casein, histone 2A and MAP2. This stimulation did not seem to be related to the cAMP binding to the R II subunit of the enzyme. Competition binding experiments showed that dipyridamole does not compete with adenosine for the A1 receptor. The results suggest that the reported regulatory properties of dipyridamole on lipid metabolism (González-Nicolás et al. Int J Biochem 21: 883-888, 1989) might be mediated through a direct action--an activation--on the catalytic subunit of a cAMP-dependent protein kinase.

Kinetics of the reaction between 5,5'-dithiobis[2-nitrobenzoic acid] and the sulphydryl group in Zn(2+)-dependent beta-lactamase II.

The kinetics of the reaction of the thiol residue in Zn(2+)-dependent beta-lactamase II with 5,5'-dithiobis[2-nitrobenzoic acid], and the concomitant inactivation revealed that both events take place at the same rate. The inactivation could not be reverted by incubation with Zn2+ or by using a substrate concentration about eight times the Km of the enzyme. EDTA incubation also produced inactivation of the enzyme, although it was reverted by increasing the substrate concentration in the assay. A dual role is proposed for Zn2+ in beta-lactamase. The kinetic analysis of the thiol modification and the concomitant inactivation is in agreement with previous reports on the implication of the metal ion in catalysis. A role in stabilizing the native structure of the enzyme is also suggested.

Degradation of beta-lactam antibiotics in the presence of Zn2+ and 2-amino-2-hydroxymethylpropane-1,3-diol (Tris). A hypothetical non-enzymic model of beta-lactamases.

The system composed of 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) and Zn2+ catalyses the degradation of cephalosporins. The beta-lactam opening fits to a first-order process, with a constant directly proportional to the zinc ion concentration. The pH and Tris concentration dependency displayed by the first-order constant, as well as the nature of the degradation products point to a mechanism that can be considered as an extension of that proposed for the benzylpenicillin degradation. The mechanism proposed here, and the values of the kinetic constants calculated, as compared with those of beta-lactamases, lead to the conclusion that the Tris-Zn2+ system simulates the catalytic action of the serine beta-lactamases rather than the action of the Zn(2+)-dependent type of enzymes.

Comparative study of various hydrogen ion buffers to assay Zn(2+)-dependent beta-lactamases.

The low Zn2+ complex formation constants, the capacity to degrade penicillin G in combination with Zn2+, and UV absorbance make 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid, 1,3-bis[tris(hydroxymethyl)methylamino]propane, and 1,4-piperazinediethanesulfonic acid convenient buffers to study Zn(2+)-dependent beta-lactamases.

Different phosphorylation behaviour of regulatory subunit isoforms of type II cAMP-dependent protein kinase from bovine heart.

Two forms of the regulatory subunit of the type II cAMP-dependent protein kinase (RII55 and RII52) were identified from bovine heart by gel electrophoretic behaviour. After autophosphorylation the RII55 isoform migrated more slowly (RII55/57) while the migration of RII52 isoform did not shift. Both isoforms showed different affinity for cAMP. The RII55/57 isoform was eluted from a cAMP-agarose column at 10 mM cAMP at low ionic strength whereas the RII52 isoform required cAMP, plus 2M NaCl. Partial proteolysis, using trypsin or formic acid, of autophosphorylated regulatory subunit isoforms resulted in different cleavage pattern as determined by peptide mapping. However, the V8 125I-peptides patterns of both isoforms are quite similar. Incubation of partially purified holoenzyme with 10 nM [gamma-32P]ATP (low ATP concentration) yielded a single band of Mr = 57,000 which corresponds to the RII55/57 isoform. The incubation, however, at 20 microM [gamma-32P]ATP yielded two phosphobands corresponding to both RII55/57 and RII52 isoforms. The phosphorylation of RII52 took place with a lower efficiency and was more sensitive to the cAMP than the corresponding phosphorylation of the RII55/57.

A stopped-flow assay for glycogen phosphorylase appropriate to measure catalytic activity at high enzyme concentrations.

Glycogen phosphorylase (EC 2.4.1.1) may be assayed in the glycogen degradation direction by a continuous spectrophotometric method. The formation of glucose 1-phosphate from glycogen and phosphate produces a controlled change of pH which can be measured by the changes in absorbance of phenol red added to the system. The procedure may be conveniently applied to a stopped-flow spectrophotometer to measure the rate of the reaction. Therefore the activity of the enzyme may be determined at low conventional concentrations and, by the same technique, at high enzyme concentrations approaching those supposed to exist in vivo.

AMP and IMP binding to glycogen phosphorylase b. A calorimetric and equilibrium dialysis study.

Reaction microcalorimetry and equilibrium dialysis have been used to study the binding of AMP and IMP to glycogen phosphorylase b (EC 2.4.1.1) at 25 degrees C and pH 6.9. The combination of both techniques has enabled us to obtain some of the thermodynamic parameters for these binding processes. Four binding sites were found to be present in the dimeric active enzyme for both AMP and IMP. The binding to two high-affinity sites, which, in our opinion, correspond to the activator sites, seems to be cooperative. The two low-affinity sites, which would then correspond to the inhibitor sites, appear to be independent when the nucleotides bind to the enzyme. The negative delta G0 of binding/site at 25 degrees C is the result in all cases of a balance between negative enthalpy and entropy changes. The large differences in delta H and delta S0 for the binding of AMP to the activator sites (-27 and -70 kJ mol-1; -22 and -150 J X K-1 mol-1) suggest the existence of rather extensive conformational changes taking place in phosphorylase b on binding with the allosteric activator. Whereas the affinity of AMP for the activator sites is about 1 order of magnitude higher than that of IMP, the affinity of both nucleotides, including their delta H and delta S0 values, seems to be the same for the inhibitor sites.